Modification of PI-TA gene conferring fungal disease resistance to plants

ABSTRACT

This invention concerns the preparation and use of an isolated nucleic acid fragments in order to confer a resistance gene mediated defense response in plants against a fungus comprising in its genome virulent and/or avirulent AVR-Pita alleles. Chimeric genes incorporating such fragments and suitable regulatory sequences can be used to create transgenic plants which can produce a resistance gene mediated defense response against a fungus comprising in its genome virulent and/or avirulent AVR-Pita alleles.

This application claims the benefit of U.S. Provisional Application No.60/248,335, filed Nov. 14, 2000, the entire contents of which are hereinincorporated by reference.

FIELD OF THE INVENTION

This invention relates to the preparation and use of an isolated nucleicacid fragment in order to confer a resistance gene mediated defenseresponse in plants against a fungus comprising in its genome virulentand/or avirulent AVR-Pita alleles. Chimeric genes incorporating suchfragments or functionally equivalent subfragments thereof and suitableregulatory sequences can be used to create transgenic plants which canproduce a resistance gene mediated defense response against a funguscomprising in its genome virulent and/or avirulent AVR-Pita alleles.

BACKGROUND OF THE INVENTION

Plants can be damaged by a wide variety of pathogenic organismsincluding viruses, bacteria, fungi and nematodes. The invasion of aplant by a potential pathogen can result in a range of outcomes: thepathogen can successfully proliferate in the host, causing associateddisease symptoms, or its growth can be halted by the host defenses. Insome plant-pathogen interactions, the visible hallmark of an activedefense response is the so-called hypersensitive response (HR). The HRinvolves rapid necrosis of cells near the site of the infection and mayinclude the formation of a visible brown fleck or lesion. Pathogenswhich elicit an HR on a given host are said to be avirulent (AVR) onthat host, the host is said to be resistant, and the plant-pathogeninteraction is said to be incompatible. Strains which proliferate andcause disease on a particular host are said to be virulent, in this casethe host is said to be susceptible, and the plant-pathogen interactionis said to be compatible.

Genetic analysis has been used to help elucidate the genetic basis ofplant-pathogen recognition for those cases in which a series of strains(races) of a particular fungal or bacterial pathogen are either virulentor avirulent on a series of cultivars of a particular host species. Inmany such cases, genetic analysis of both the host and the pathogenrevealed that many avirulent fungal and bacterial strains differ fromvirulent ones by the possession of one or more avirulence (“avr” or“AVR”) genes that have corresponding “resistance” (R) genes in the host.

This avirulence gene-resistance gene model is termed the “gene-for-gene”model (Crute et al. (1985) pp 197-309 in: Mechanisms of Resistance ofPlant Disease. R. S. S. Fraser, ed.; Ellingboe, (1981) Annu. Rev.Phytopathol. 19:125-143; Flor, (1971) Annu. Rev. Phythopathol.9:275-296). According to a simple formulation of this model, plantresistance genes encode specific receptors for molecular signalsgenerated by avr genes. Signal transduction pathway(s) then carry thesignal to a set of target genes that initiate the host defenses. Despitethis simple predictive model, the molecular basis of the avr-resistancegene interaction is still unknown.

The first R-gene cloned was the Hm1 gene from corn (Zea mays), whichconfers resistance to specific races of the fungal pathogen Cochlioboluscarbonum (Johal et al., 1992, Science 258:985-987). Hm1 encodes areductase that detoxifies a toxin produced by the pathogen. Next to becloned was the Pto gene from tomato (Lycopersicon pimpinellifollium)(Martin et al., 1993, Science 262:1432-1436; U.S. Pat. No. 5,648,599).Pto encodes a serine-threonine protein kinase that confers resistance intomato to strains of the bacterial pathogen Pseudomonas syringae pv.tomato that express the avrPto avirulence gene. Taking center stage noware R-genes that encode proteins containing leucine-rich-repeats (LRRs)(Jones and Jones, 1997, Adv. Bot Res. Incorp. Adv. Plant Pathol.24:89-167). Two classes of membrane anchored proteins with extracellularLRRs have been identified. One subclass includes R-gene products thatlack a cytoplasmic serine/threonine kinase domain such as the tomatoCf-9 gene for resistance to the fungus Cladosporium fulvum (Jones etal., 1994, Science 266:789-793, WO 95/18230), and the other subclassincludes an R-gene product with a cytoplasmic serine/threonine kinasedomain, the rice Xa-21 gene for resistance to the bacterial pathogenXanthomonas oryzae (Song et al., 1995, Science 270:1804-1806; U.S. Pat.No. 5,859,339). The largest class of R-genes includes those encodingproteins with cytoplasmic LRRs such as the Arabidopsis R-genes RPS2(Bent et al., 1994, Science 265:1856-1860; Mindrinos et al., 1994, Cell78:1089-1099) and RPM1 (Grant et al., 1995, Science 269:843-846). TheseR-proteins also possess a putative nucleotide binding site (NBS), andeither a leucine zipper (LZ) motif or a sequence homologous to theToll/Interleukin-1 receptor (TIR). Table 1 has been reproduced in partfrom Baker et al. (1997, Science 276:726-733) as a concise summary ofclasses of R-genes cloned to date and examples of cloned genes withineach class.

TABLE 1 Isolated plant resistance genes¹ Class R gene Plant PathogenStructure 1 RPS2 Arabidopsis Pseudomonas syringae LZ-NBS-LRR pv. TomatoRPM1 Arabidopsis P. syringae pv. LZ-NBS-LRR Maculicola Prf Tomato P.syringae pv. Tomato LZ-NBS-LRR N Tobacco Tobacco mosaic virusTIR-NBS-LRR L⁶ Flax Melampsora lini TIR-NBS-LRR M Flax M. liniTIR-NBS-LRR RPP5 Arabidopsis Peronospora parasitica TIR-NBS-LRR I₂Tomato Fusarium oxysporum NBS-LRR 2 Pto Tomato P. syringae pv. TomatoProtein kinase 3 Cf-9 Tomato Cladosporium fulvum LRR-TM Cf-2 Tomato C.fulvum LRR-TM HS1^(pro-1) Sugar beet Heterodera schachtii LRR-TM 4 Xa21Rice Xanthomonas oryzae LRR, protein kinase pv. Oryzae 5 Hm1 MaizeCochilobolus carbonum, Toxin race 1 reductase ¹This Table has beenreproduced in part from Baker et al. (1997, Science 276:726-733).References for each of the listed R-genes can be found in this reviewarticle.

Nucleotide binding sites (NBS) are found in many families of proteinsthat are critical for fundamental eukaryotic cellular functions such ascell growth, differentiation, cytoskeletal organization, vesicletransport, and defense. Key examples include the RAS group, adenosinetriphosphatases, elongation factors, and heterotrimeric GTP bindingproteins called G-proteins (Saraste et al., 1990, Trends in Biochem.Science 15:430). These proteins have in common the ability to bind ATPor GTP (Traut, 1994, Eur. J. Biochem. 229:9-19).

It has long been hypothesized that the rice blast system represented aclassical gene-for-gene system as defined by H. H. Flor (Flor, 1971,Annu. Rev. Phytopathol. 19:125-143). Genetic analyses needed to identifyAVR-genes in the rice blast pathogen, Magnaporthe grisea, has beenhampered by the low fertility that typifies M. grisea field isolatesthat infect rice. Genetic crosses between poorly fertile M. grisea ricepathogens and highly fertile M. grisea pathogens of other grasses (suchas weeping lovegrass, Eragrostis curvula, and finger millet, Eleusinecoracana) have provided laboratory strains of the fungus with the levelof sexual fertility required for identifying AVR-genes (Valent et al.,1991, Genetics 87-101). Rare fertile rice pathogens have since alloweddemonstration of a one-to-one genetic or functional correspondencebetween blast fungus AVR-genes and particular rice R-genes (Silue etal., 1992, Phytopathology 82:577-580).

Interest in the rice blast pathosystem is keen because rice blastdisease, caused world-wide by the fungal pathogen Magnaporthe grisea(Hebert) Barr (anamorph Pyricularia grisea Sacc.), continues as the mostexplosive and potentially damaging disease of the rice crop despitedecades of research towards its control. Manipulation of blastresistance genes remains one of the primary targets in all rice breedingprograms, as fungal populations evolve to defeat deployed resistancestrategies (See The Rice Blast Disease, 1994, ed. Zeigler, Leong andTeng, CAB International, Wallingford).

Commercial fungicide usage to supplement genetic control strategiesbegan around 1915 when rice farmers used inorganic copper-basedfungicides (Chapter 29 in The Rice Blast Disease, 1994, ed. Zeigler,Leong and Teng, CAB International, Wallingford). The fungicides used tocontrol blast disease have changed through time, with some compounds,such as the organomercurials used in the 1950s, causing majorenvironmental damage. The control of rice blast with fungicidescurrently represents a cost of more than $500 million per year tofarmers. This expense for blast control is the largest segment of theworld rice fungicide market, which totaled $752 million in 1998 (WoodMackenzie). Expectations are that the disease problems will intensify asthe world rice requirements increase by an estimated 1.7% annuallybetween 1990 and 2025 (See The Rice Blast Disease, 1994, ed. Zeigler,Leong and Teng, CAB International, Wallingford). This estimated need foran additional 13 million tons of rough rice per year to feed the growingpopulation must come from intensification of production on decreasingavailable land. Rice blast disease is favored by agronomic productionpractices aimed at high yields, and thus the disease will continue, andmost likely increase, as a constraint to rice crop yields unless durablegenetic resistance against rice blast disease can be engineered intorice. This invention represents an advance towards the long term goal ofengineering durable genetic resistance to rice blast by generating novelPi-ta alleles that have different specificities as regards the spectrumof AVR-Pita gene products they recognize. The fungus M. grisea has alarge host range including species of different tribes within the grassfamily, Triticeae (e.g., wheat), Oryzeae (e.g., rice), Clorideae (e.g.,finger millet), Paniceae (e.g., pearl millet), Andropogoneae (e.g.,sorghum) and Maydeae (e.g., maize). Molecular analyses have now defined8 host species-specific subpopulations of M. grisea, each with arestricted set of host species specificities (Reviewed by Valent, 1997,The Mycota V, Plant Relationships, Carroll/Tudzynski, eds.,Springer-Verlag Berlin Heidelberg pp 37-54). Table 2 gives a currentview of pathogen subpopulations according to mitochondrial DNA (mtDNA)type. This view is strongly supported by separate analyses of ribosomalDNA (rDNA) polymorphisms (including both Restriction Fragment LengthPolymorphism (RFLP) and Internal Transcribed Spacer (ITS) sequences) andof polymorphisms in both repetitive DNAs and single copy sequences.

The pathogens of rice, wheat, finger millet, barley and corn (mtDNAtypes Ia-e) appear closely related, while pathogens of Digitaria spp.and Pennisetum spp. (mtDNA types II-IV) are highly divergent from theprevious groups and from each other. However, M. grisea strainsthroughout this broad host range can cause significant crop damage. Thispathogen has been shown to be the main cause of yield loss of fingermillet (Eleusine coracana) in Africa, while infections in wheat(Triticum aestivum; Urashima et al., 1993, Plant Disease 77:1211-1216)and pearl millet (Pennisetum glaucum; Hanna et al., 1989, J. Heredity80:145-147), although less widespread, can be severe under humid weatherconditions. The disease has been documented on barley and corn (Seerefs. In Urashima et al., 1993, Plant Disease 77:1211-1216).

TABLE 2 Host Specificities Within Magnaporthe grisea Subpopulation¹Defining Host Species² Crops at Risk Ia Oryza sativa Rice, Barley, CornIb Triticum aestivum Wheat, Barley Ie Eleusine spp. Finger Millet IcEleusine spp. Finger Millet IIa Digitaria spp. IIb Digitaria spp. IIIPennisetum spp. Pearl Millet IV Pennisetum spp. ¹Designated bymitochondrial-DNA haplotype. ²Pathogenicity to the “Defining HostSpecies” appears conserved within the subpopulation. Some hosts, such asbarley, are infected by members of two or more subpopulations.

Knowledge of pathogenicity and host specificity for plant pathogenicfungi is not as advanced as for bacterial and viral pathogens, an dlikewise, less is known about the molecular basis of resistance incereal crop plants than in dicot crops or in dicot model systems such asArabidopsis (Baker et al., 1997, Science 276:726-733). Sasaki reportedthe first results on the inheritance of resistance to rice blast diseasefrom studies begun in Japan in 1917 (Sasaki, 1922, Japanese Genetics,Japan 1, 81-85).

Since this time, over 30 R-genes have been defined through extensivegenetic analysis worldwide, and many of these blast resistance geneshave been mapped to rice chromosomes (See Refs. In Takahashi, 1965, TheRice Blast Disease, Johns Hopkins Press, Baltimore, 303-329; Causse etal., 1994, Genetics 138:1251-1274). These R-genes include 20 majorresistance genes and 10 putative quantitative trait loci (QTLs).Kiyosawa has described 13 major resistance genes with 9 of these genesfound as multiple alleles at 3 loci; 5 at the Pi-k locus on chromosome11, 2 at the Pi-z locus on chromosome 6 and 2 at the Pi-ta locus onchromosome 12 (Kiyosawa, 1984, Rice Genetics Newsletter 1:95-97). Recentstudies in Japan (Ise, 1992, International Rice Research Newsletter17:8-9) and at the International Rice Research Institiute (IRRI)(Mackill et al., 1992, Phytopathology 82:746-749) have produced nearisogenic rice lines (NILs) for use as “differential” rice varieties fordetermining which resistance genes are effective in controllingindividual strains of the fungus. The IRRI NILs, which provide indicadifferentials for the blast fungus populations in tropical regions, havebeen analyzed for genetic relationships between their resistance genesand those present in Kiyosawa Differentials (Inukai et al., 1994,Phytopathology 84:1278-1283).

Molecular markers (or “tags”) tightly linked to R-genes have utility forefficient introgression and manipulation of those R-genes in breedingprograms. By comparing genotypic patterns of near-isogenic lines, theirdonors, and their recurrent parents, Yu et al. (1987, Phytopathology77:323-326) were able to identify five restriction fragment lengthpolymorphic (RFLP) markers linked to three blast resistance genes and tomap them to rice chromosomes using segregating populations. RFLP markerslinked to the R-genes have been reported (Yu et al., 1991, Theor ApplGenet 81:471-476). Molecular cloning of agronomically important R-genesrepresents a further advance to the ability of researchers to combineR-genes with other input and output traits in key crop varieties.

In the course of the above mentioned investigations on the inheritanceof resistance, Sasaki discovered physiological races of the rice blastpathogen by observing that different field isolates of the blast fungusvary in their ability to cause disease on different varieties of rice(Sasaki, 1922, Journal of Plant Protection 9:631-644; Sasaki, 1923,Journal of Plant Protection 10:1-10). Instability, or “breaking down”under field conditions, of major R-gene resistance to the rice blastfungus has resulted in identification of numerous races, or pathotypes,defined according to virulence spectra on differential rice varieties(Chapters 13 and 16 in The Rice Blast Disease, 1994, ed. Zeigler, Leongand Teng, CAB International, Wallingford). Pathogen populations aredynamic in response to deployment of a new resistance gene, sometimesresulting in new races that overcome the resistance gene within one ortwo years after deployment in the field.

Accordingly, incorporation of diseases resistance (R) genes into cropplants has not achieved durable resistance to highly variable fungalpathogens such as Magnaporthe grisea (Hebert) Barr, the causal agent ofthe devastating rice blast disease worldwide. (Rossman et al.,Commonwealth Mycological Institute, Kew, Surrey, Second Edition, 1985;Rice Blast Disease, Zeigler et al., eds., CAB International,Wallingford, Oxon OX108DE, UK (1994)). In other words, R-gene utility incontrolling rice blast disease has bee n limited by the inherent fieldvariability of the pathogen.

There are a number of virulent AVR-Pita alleles in different strains ofM. grisea for which no corresponding R-gene variants have beenidentified in rice that recognize these alleles. No one heretofore hasbeen able to engineer an R-gene to recognize such alleles. Clearly, anability to do so would provide a valuable tool to control currentlyvirulent strains of the rice blast fungus and other pathogens.

Clearly, researchers have not adequately succeeded in this regard.

Applicants' assignee's copending patent application which was filed onJun. 21, 1999 and having application Ser. No. 09/336, 946 (PCTPublication No. WO 00/08162, which was published on Feb. 17, 2000),describes a Pi-ta gene conferring disease resistance. It does notaddress the need to modify R-genes to increase their utility by alteringtheir specificity with respect to the AVR-Pita alleles which it canrecognize in different strains of a fungus.

Wang et al. (1999) Plant J 19:55-64 describe another rice blastresistance gene, Pib, different from Pi-ta.

WO 00/34479, which published on Jun. 15, 2000, describes nucleic acidfragments which encode a different disease resistance protein thatconfers resistance to M. grisea.

U.S. Pat. No. 5,648,599, issued to Tanksley and Martin on Jul. 15, 1997,describes an isolated gene fragment from tomato which encodes the Ptoserine/threonine kinase, conferring disease resistance to plants byresponding to an avirulence gene in a bacterial plant pathogen.

WO 95/28423, which published on Oct. 26, 1995, describes resistance dueto the Pseudomonas syringae RPS2 gene family, primers, probes anddetection methods. This published international application includesbroad claims to genes encoding proteins with particular NH₂-terminalmotifs, NBS motifs and leucine rich repeats for protecting plantsagainst pathogens. There are some unique features of the Pi-ta protein.The Pi-ta gene product has a unique amino terminus, lacking either thepotential leucine zipper motif of the RPS2 gene-product subfamily (Bentet al., 1994, Science 265:1856-1860; Mindrinos et al., 1994, Cell78:1089-1099) or the Toll/Interleukin-1 receptor homology encoded by theN gene subfamily (Whitman et al., 1994, Cell 78:1101-1115). Mostimportantly, the carboxy terminal portion of the Pi-ta gene product isleucine rich, but it does not fit the consensus sequences forleucine-rich repeats reported for R-gene products (Jones and Jones,1997, Adv. Bot Res. Incorp. Adv. Plant Pathol. 24:89-167).

U.S. Pat. No. 5,571,706, issued to Baker et al. on Nov. 5, 1996, coversplant virus resistance conferred by the N gene.

U.S. Pat. No. 5,859,351, issued to Staskawicz et al. on Jan. 12, 1999,describes the PRF protein and nucleic acid sequence, which is involvedin disease resistance in tomato.

U.S. Pat. No. 5,859,339, issued to Ronald et al. on Jan. 12, 1999,describes the first resistance gene cloned from rice, Xa-21, whichencodes an integral membrane protein with both LRR and serine/threoninekinase domains, and confers resistance in rice to bacterial blight.

WO 91/15585 which published on Oct. 17, 1991 and U.S. Pat. No. 5,866,776issued to de Wit et al. on Feb. 2, 1999 describe a method for theprotection of plants against pathogens using a combination of a pathogenavirulence gene and a corresponding plant resistance gene.

U.S. Pat. No. 5,674,993 ('993 patent), issued to Kawasaki et al. on Oct.7, 1997, describes nucleic acid markers that co-segregate with the riceblast resistance genes Pi-b, Pi-ta and Pi-ta² and the suggestion thatrice blast resistance genes could be isolated and cloned by using thesenucleic acid markers. However, no nucleotide sequences are provided forany rice blast resistance genes in the '993 patent. It should be notedthat a putative sequence for the Pi-b rice blast resistance gene is nowavailable in Genbank (accession number AB013448).

In addition, Kawasaki et al. have also published two papers. The firstpaper, Rybka et al., MPMI, 10(4):517-524 (1997), is entitled “HighResolution Mapping of the Indica-Derived Rice Blast Resistance Genes.II. Pi-ta² and Pi-ta and a Consideration of Their Origin.” The sequencefor the RAPD primer that is set forth at the top of column 2 on page 519is not the same as the RAPD primer set forth in SEQ ID NO:2 in the '993patent. It is not clear which sequence is correct. Notwithstanding this,it is clear that this paper does not set forth any nucleotide sequencesfor any rice blast genes. The second paper is Nakamura et al., Mol. Gen.Genet. 254:611-62 (1997). This paper describes the construction of an800-kb contig in the near-centromeric region of the rice blastresistance gene Pi-ta² using a rice BAC library. Again, no nucleotidesequence for any rice blast genes is disclosed.

Thus, it is believed that no one heretofore has addressed the need tomodify R-genes to increase their utility by broadening their specificitywith respect to the AVR-Pita alleles. The broadened specificity enablesthe modified R-gene to recognize different fungal strains.

SUMMARY OF THE INVENTION

This invention relates to an isolated nucleic acid fragment comprising anucleic acid sequence or subsequence thereof encoding an altered Pi-taresistance polypeptide wherein the polypeptide has a single amino acidalteration at position 918 which confers a resistance gene mediateddefense response against a fungus comprising in its genome virulentand/or avirulent AVR-Pita alleles.

In another aspect, this invention concerns alterations at position 918which are selected from the group consisting of M, C, I, R, K, N, L andQ.

In still another aspect, this invention concerns chimeric genescomprising the nucleic acid fragment of the invention.

Also of interest are plants comprising in their genome the chimericgenes described herein as well as seeds obtained from such plants.

In an even further aspect, this invention concerns a method ofconferring a resistance gene mediated defense response in plants againsta fungus comprising in its genome virulent and/or avirulent AVR-Pitaalleles in plants which comprises:

(a) transforming a plant with a chimeric gene of the invention; and

(b) selecting transformed plants of step (a) which are resistant to afungus comprising in it genome virulent and/or avirulent AVR-Pitaalleles.

Biological Deposit

The fungal strain O-137 (collected in 1985 at the China National RiceResearch Institute in Hangzhou) has been deposited with the AmericanType Culture Collection (ATCC), 10801 University Boulevard, Manassas,Va. 20110-2209, and bears the following designation, accession numberand date of deposit. Fungal strain G-213, a pathogen isolated fromDigitaria smutsii in Japan, was obtained from the collection of JeanLoup Notteghem, Laboratoire de phytopathologie, Institut de RecherchesAgronomiques Tropicales et des Cultures Vivrieres, Centre de CooperationInternationale en Recherche Agronomique pour le Developpement (CIRAD),BP 5035, 34032 Montpellier Cedex 1, France, and is available under thename JP34. Rice pathogen strain G-198, also obtained from Jean LoupNotteghem, was originally isolated from barley in Thailand. Ricepathogen strain GUY11, also obtained from Jean Loup Notteghem, isdescribed in Leung et al. (1988) Phytopathology 78, 1227-1233. Ricepathogen strain Ina 72 is described in Kiyosawa (1976) SABRAO Journal8:53-67. All strains have been deposited with the ATCC.

Plasmid pCB2022 which contains sequences of Pi-ta promoter, Pi-ta cDNA,linker sequence and In2-1 terminator sequence described in Example 6 haslikewise been deposited with the ATCC.

Designation Material Accession Number Date of Deposit O-137 M. griseaATCC 74457 Aug. 3, 1998 G-213 M. grisea PTA-191  Jun. 8, 1999 G-198 M.grisea PTA-190  Jun. 8, 1999 GUY 11 M. grisea PTA-192  Jun. 8, 1999 Ina72 M. grisea PTA-2606 Oct. 18, 2000 pCB2022 Plasmid PTA-2631 Oct. 25,2000

BRIEF DESCRIPTION OF THE SEQUENCE LISTINGS AND FIGURES

SEQ ID NO:1 sets forth the sequence of plasmid pCB980 which is a 4119 bpplasmid containing sequences of pBluescript SK+ (nucleotides 1-676,1880-4119), AVR-Pita promoter (nucleotides 677-1157) and AVR-Pita cDNA(nucleotides 1158-1829) from M. grisea strain O-137.

SEQ ID NO:2 is the 672 nucleotide sequence of an AVR-Pita cDNA from M.grisea strain O-137.

SEQ ID NO:3 is the predicted amino acid sequence of the protein encodedby the AVR-Pita cDNA sequence set forth in SEQ ID NO:2.

SEQ ID NO:4 is the 788 nucleotide sequence of an AVR-Pita cDNA from M.grisea strain G-198.

SEQ ID NO:5 is the predicted amino acid sequence of the protein encodedby the AVR-Pita cDNA set forth in SEQ ID NO:4.

SEQ ID NO:6 is a predicted 672 nucleotide sequence of an AVR-Pita cDNAfrom M. grisea strain GUY 11 based on genomic sequence set forth in SEQID NO:59.

SEQ ID NO:7 is the predicted amino acid sequence of the protein encodedby the AVR-Pita cDNA set forth in SEQ ID NO:6.

SEQ ID NO:8 is the 884 nucleotide sequence of an AVR-Pita genomic clonefrom M. grisea strain Ina 72.

SEQ ID NO:9 is a predicted 675 nucleotide sequence of an AVR-Pita cDNAfrom M. grisea strain Ina 72 based on genomic sequence set forth in SEQID NO:8.

SEQ ID NO:10 is the predicted amino acid sequence of the protein (Ina72.1) encoded by the predicted AVR-Pita cDNA sequence set forth in SEQID NO:9.

SEQ ID NO:11 is the 884 nucleotide sequence of a second AVR-Pita genomicclone from M. grisea strain Ina 72.

SEQ ID NO:12 is a predicted 675 nucleotide sequence of an AVR-Pita cDNAfrom M. grisea strain Ina 72 based on genomic sequence set forth in SEQID NO:11.

SEQ ID NO:13 is the predicted amino acid sequence of the protein (Ina72.2) encoded by the predicted AVR-Pita cDNA sequence set forth in SEQID NO:12.

SEQ ID NO:14 is the 884 nucleotide sequence of an AVR-Pita genomic clonefrom M. grisea strain G-213.

SEQ ID NO:15 is a predicted 675 nucleotide sequence of an AVR-Pita cDNAfrom M. grisea strain G-213 based on genomic sequence set forth in SEQID NO:14.

SEQ ID NO:16 is the predicted amino acid sequence of the protein encodedby the predicted AVR-Pita cDNA sequence set forth in SEQ ID NO:15.

SEQ ID NOS:17 and 18 are PCR primers used to amplify AVR-Pita cDNA andgenomic DNA from strain O-137, and an AVR-Pita genomic nucleic acidfragment from strains G-198 and GUY11.

SEQ ID NOS:19 and 20 are PCR primers used to amplify a functionalAVR-Pita promoter fragment from strain O-137.

SEQ ID NO:21 is a PCR primer used along with SEQ ID NO:18 to amplifyAVR-Pita genomic nucleic acid fragments from strain Ina 72.

SEQ ID NOS:22 and 23 are PCR primers used to amplify an AVR-Pita genomicnucleic acid fragment from strain G-213.

SEQ ID NOS:24 and 25 are PCR primers used to amplify an AVR-Pita nucleicacid fragment in the process of constructing pAVR3.

SEQ ID NOS:26 and 27 are PCR primers used to amplify AVR-Pita₁₇₆, anAVR-Pita nucleic acid fragment that directly encodes the putative matureprotease.

SEQ ID NOS:28 and 29 are PCR primers used to amplify AVR-Pita cDNA fromstrain G-198.

SEQ ID NOS:30 and 31 are PCR primers used to amplify a partial Pi-tacDNA.

SEQ ID NO:32 is a PCR primer used along with SEQ ID NO:30 to amplify asusceptible Pi-ta nucleic acid fragment from susceptible C101A51 rice.

SEQ ID NOS:33-51 are PCR primers used to modify Pi-ta coding sequence,resulting in an array of plasmids comprising nucleic acid fragments thatencode different Pi-ta proteins with all 20 amino acids represented atposition 918.

SEQ ID NOS:52 and 53 are PCR primers used to amplify the Pi-ta leucinerich domain (LRD).

SEQ ID NOS:54 and 55 are PCR primers used to amplify the In2-1terminator sequence.

SEQ ID NO:56 is the 5757 nucleotide sequence of the genomic clone of thePi-ta gene from Oryza sativa variety Yashiro-mochi.

SEQ ID NO:57 is the 5222 nucleotide sequence of an EcoRI-Hind IIIfragment that contains 2425 bp of the native Pi-ta promoter (nucleotides1 to 2425) and Pi-ta cDNA (nucleotides 2426-5212) from rice varietyYashiro-mochi.

SEQ ID NO:58 is the predicted Pi-ta protein sequence encoded by thePi-ta nucleotide sequences set forth in SEQ ID NOS:56 and 57.

SEQ ID NO:59 is the 881 nucleotide sequence of an AVR-Pita genomic clonefrom M. grisea strain GUY11.

SEQ ID NO:60 sets forth the sequence of the insert in plasmid pCB2022which contains sequences of Pi-ta promoter (nucleotides 1-2425), Pi-tacDNA (nucleotides 2426-5212), linker sequence (nucleotides 5213-5241)and In2-1 terminator sequence (nucleotides 5242-5690).

FIG. 1. Comparison of the deduced amino acid sequences encoded byavirulent and virulent AVR-Pita nucleic acid fragments. The AVR-Pitanucleic acid fragments from avirulent strains O-137 (SEQ ID NO:3) andG-213 (SEQ ID NO:16), and the first sequence from strain Ina 72 (Ina72.1; SEQ ID NO:10) have been shown to confer avirulence (Example 5).The nucleic acid fragment encoding the second Ina 72 sequence (Ina 72.2;SEQ ID NO:13) and the fragments from virulent strains G-198 (SEQ IDNO:5) and Guy11 (SEQ ID NO:7) do not confer avirulence (Example 5). Onlydifferences from the deduced amino acid sequence of AVR-Pita from strainO-137 (SEQ ID NO:3) are indicated. Identical amino acids are indicatedby a dash (-), whereas a gap that is introduced to maximize alignment isindicated by a dot (.). The underlined amino acids indicate the putativeprotease motif.

FIG. 2. Diagram of constructs in plasmids pCB1947, pML63 and pCB1926.

A. pCB1947 contains a construct of the AVR-Pita isolated nucleic acidfragment (formerly called AVR2-YAMO) engineered to encode directly theputative mature protease for the transient expression experiments. Thisprocessed form of the isolated nucleic acid fragment, designatedAVR-Pita₁₇₆, encodes a polypeptide comprised of amino acids 48 to 223 asset forth in SEQ ID NO:6. An initiation methionine was added to theconstruct through the NcoI site used in the cloning process. TheAVR-Pita coding sequence was first amplified by PCR using primers YL30(SEQ ID NO:26) containing an in-frame NcoI site and YL37 (SEQ ID NO:27)containing a KpnI site, and cloned into the NcoI-KpnI site of pML142,resulting in vector pCB1947. The maize Adh1-6 intron inserted downstreamof the 35S promoter results in enhanced expression in monocots. Thisintron is described in Mascarenhas et al. (1990) Plant Mol Biol.15:913-920.

B. pML63 contains the uidA gene (which encodes the GUS enzyme) operablylinked to the CaMV35S promoter and 3′ NOS sequence. pML63 is modifiedfrom pMH40 to produce a minimal 3′ NOS terminator fragment. pMH 40 isdescribed in WO 98/16650 which published on Apr. 23, 1998, thedisclosure of which is hereby incorporated by reference. Using standardtechniques familiar to those skilled in the art, the 770 base pairterminator sequence contained in pMH40 was replaced with a new 3′ NOSterminator sequence comprising nucleotides 1277 to 1556 of the sequencepublished by Depicker et al. (1982, J. Appl. Genet. 1:561-574).

C. pCB1926 contains the Pi-ta cDNA construct that was created by firstamplifying a 2.1 kb partial Pi-ta cDNA nucleic acid fragment from firststrand cDNA using primers F12-1 (SEQ ID NO:31) and GB67 (SEQ ID NO:30).A synthetic full-length cDNA was generated by incorporating a 706 bpNcoI-BamHI fragment containing the 5′ end of the genomic Pi-ta gene frompCB1649, resulting in plasmid pCB1906. A 3.1 kb EcoRI fragment frompCB1649 containing 2425 bp of the native Pi-ta promoter sequences(pPi-ta) and 736 bp of the 5′ Pi-ta coding sequence was then insertedinto the EcoRI sites of pCB1906 to replace the 736 bp 5′ end of thesynthetic cDNA nucleic acid fragment, producing pCB1926.

FIG. 3. DNA Genomic Blot Analysis of AVR-Pita Copy Number. M. griseagenomic DNA from the indicated strains was isolated as describedpreviously (Sweigard et al., 1995, The Plant Cell 7:1221-1233), digestedwith Eco RI, electrophoretically fractionated through an agarose gel,blotted onto a filter, and probed with an AVR-Pita fragment usingstandard molecular biology protocols (Sambrook). Since there is no EcoRI site in the AVR-Pita gene, the number of bands per lane indicates thenumber of AVR-Pita genes present in that particular M. grisea strain.For example, the leftmost lane shows the result for M. grisea strainG-198 which shows two bands, indicating that G-198 has two AVR-Pitagenes. The short horizontal lines along the left border of the figureindicate the location of DNA size markers from a HindIII digest of λ DNAof sizes from top to bottom of 23.1 kb, 9.4 kb, 6.6 kb, 4.4 kb, 2.3, and2.0 kb.

DESCRIPTION OF THE INVENTION

In the context of this disclosure, a number of terms shall be utilized.

The term “disease resistance gene” means a gene encoding a polypeptidecapable of triggering a defense response in a plant cell or planttissue. The terms “disease resistance gene”, “resistance (R) gene” and“R” gene are used interchangeably herein. The resistance that resultsfrom a defense response can take several forms. For example, in somegenetic backgrounds resistance may take the form of no visible symptomson inoculated plant tissue, and in others resistance may include smallbrown spots from which the fungus does not sporulate and does notreinitiate infection. In both cases, the fungal pathogen does notcomplete its life cycle and disease development is stopped. In somecases disease resistance may take the form of smaller-sized lesions thatdo not produce the quantity of fungal spores typical of the fulldisease.

A “defense response” is a specific defensive reaction produced by ahost, e.g., a plant, to combat the presence of an infectious agent orpathogen.

A “Pi-ta resistance gene” is a disease resistance gene encoding aPi-ta-resistance polypeptide capable of triggering a defense response ina plant cell or plant tissue against a fungal pathogen such asMagnaporthe grisea.

The term “Pi-ta resistance gene mediated defense response” means adefense response due to the production of the polypeptide encoded by thePi-ta resistance gene and elicited by the presence of a fungal pathogen.The term “resistance gene mediated defense response” means a defenseresponse due to the production of a polypeptide encoded by a resistancegene and elicited by the presence of a fungal pathogen or a fungalpathogen elicitor.

A “fungal pathogen elicitor” is a pathogen signal molecule that isdirectly or indirectly recognized by a resistance gene product.

A “virulent AVR-Pita allele” is a variant of the AVR-Pita gene whosegene product normally does not elicit a Pi-ta resistance gene-mediateddefense response in rice that expresses the functional Pi-ta resistanceprotein (A918 described herein; SEQ ID NO:58) such as Yashiro-mochi. Theavr-pita gene from M. grisea strain G-198 described herein is an exampleof a virulent AVR-Pita allele.

An “avirulent AVR-Pita allele” is a variant of the AVR-Pita gene whosegene product normally elicits a Pi-ta resistance gene-mediated defenseresponse in rice that expresses the functional Pi-ta resistance protein(A918 described herein; SEQ ID NO:58) such as Yashiro-mochi. TheAVR-Pita gene from M. grisea strain O-137 described herein is an exampleof an avirulent AVR-Pita allele.

An “AVR-Pita isolated nucleic acid fragment” is a nucleic acid fragmentisolated from a pathogen wherein the nucleic acid fragment encodes apolypeptide whose direct or indirect interaction with the Pi-taresistance protein is responsible for triggering the Pi-ta resistancegene mediated defense response.

An “isolated nucleic acid fragment” is a polymer of RNA or DNA that issingle- or double-stranded, optionally containing synthetic, non-naturalor altered nucleotide bases. An isolated nucleic acid fragment in theform of a polymer of DNA may be comprised of one or more segments ofcDNA, genomic DNA or synthetic DNA.

The terms “subfragment that is functionally equivalent” “functionallyequivalent subfragment”, “functionally equivalent subsequence” and“subsequence” are used interchangeably herein. These terms refer to aportion or subsequence of an isolated nucleic acid fragment in which theability to alter gene expression or produce a certain phenotype isretained whether or not the fragment or subfragment encodes an activeenzyme. For example, the fragment or subfragment can be used in thedesign of chimeric genes to produce the desired phenotype in atransformed plant. Chimeric genes can be designed for use inco-suppression or antisense by linking a nucleic acid fragment orsubfragment thereof, whether or not it encodes an active enzyme, in theappropropriate orientation relative to a plant promoter sequence.

The terms “substantially similar” and “corresponding substantially” asused herein refer to nucleic acid fragments wherein changes in one ormore nucleotide bases does not affect the ability of the nucleic acidfragment to mediate gene expression or produce a certain phenotype.These terms also refer to modifications of the nucleic acid fragments ofthe instant invention such as deletion or insertion of one or morenucleotides that do not substantially alter the functional properties ofthe resulting nucleic acid fragment relative to the initial, unmodifiedfragment. It is therefore understood, as those skilled in the art willappreciate, that the invention encompasses more than the specificexemplary sequences.

Moreover, the skilled artisan recognizes that substantially similarnucleic acid sequences encompassed by this invention are also defined bytheir ability to hybridize, under moderately stringent conditions (forexample, 0.5×SSC, 0.1% SDS, 60° C.) with the sequences exemplifiedherein, or to any portion of the nucleotide sequences reported hereinand which are functionally equivalent to the isolated nucleic acidfragment of the invention. Preferred substantially similar nucleic acidsequences encompassed by this invention are those sequences that are 80%identical to the nucleic acid fragments reported herein or which are 80%identical to any portion of the nucleotide sequences reported herein.More preferred are nucleic acid fragments which are 90% identical to thenucleic acid sequences reported herein, or which are 90% identical toany portion of the nucleotide sequences reported herein. Most preferredare nucleic acid fragments which are 95% identical to the nucleic acidsequences reported herein, or which are 95% identical to any portion ofthe nucleotide sequences reported herein. Sequence alignments andpercent similarity calculations may be determined using the Megalignprogram of the LASARGENE bioinformatics computing suite (DNASTAR Inc.,Madison, Wis.). Multiple alignment of the sequences are performed usingthe Clustal method of alignment (Higgins and Sharp (1989) CABIOS.5:151-153) with the default parameters (GAP PENALTY=10, GAP LENGTHPENALTY=10). Default parameters for pairwise alignments and calculationof percent identiy of protein sequences using the Clustal method areKTUPLE=1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5. For nucleicacids these parameters are GAP PENALTY=-10, GAP LENGTH PENALTY=10,KTUPLE=2, GAP PENALTY=5, WINDOW=4 and DIAGONALS SAVED=4. A “substantialportion” of an amino acid or nucleotide sequence comprises enough of theamino acid sequence of a polypeptide or the nucleotide sequence of agene to afford putative identification of that polypeptide or gene,either by manual evaluation of the sequence by one skilled in the art,or by computer-automated sequence comparison and identification usingalgorithms such as BLAST (Altschul, S. F., et al., (1993) J. Mol. Biol.215:403-410) and Gapped Blast (Altschul, S. F. et al., (1997) NucleicAcids Res. 25:3389-3402).

“Gene” refers to a nucleic acid fragment that expresses a functional RNAtranscript, such as but not limited to mRNA, rRNA, tRNA, or antisenseRNA, including regulatory sequences preceding (5′ non-coding sequences)and following (3′ non-coding sequences) the coding sequence. “Nativegene” refers to a gene as found in nature with its own regulatorysequences. “Chimeric gene” refers to any gene that is not a native gene,comprising at least one regulatory sequence and coding sequence that arenot found together in nature. Accordingly, a chimeric gene may compriseregulatory sequences and coding sequences that are derived fromdifferent sources, or regulatory sequences and coding sequences derivedfrom the same source, but arranged in a manner different than that foundin nature. “Endogenous gene” refers to a native gene in its naturallocation in the genome of an organism. A “foreign” gene refers to a genenot normally found in the host organism, but that is introduced into thehost organism by gene transfer. Foreign genes can comprise native genesinserted into a non-native organism, or chimeric genes. A “transgene” isa gene that has been introduced into the genome by a transformationprocedure.

“Coding sequence” refers to a DNA sequence that codes for an RNAtranscript, and in the case of a gene encoding a polypeptide, a specificamino acid sequence. “Regulatory sequences” refer to nucleotidesequences located upstream (5′ non-coding sequences), within, ordownstream (3′ non-coding sequences) of a coding sequence, and whichinfluence the transcription, RNA processing or stability, or translationof the associated coding sequence. Such sequences can be native ornon-native. Regulatory sequences may include, but are not limited to,promoters, translation leader sequences, introns, and polyadenylationrecognition sequences.

“Pathogen” refers to an organism or an infectious agent whose infectionaround or inside the cells of viable plant tissue elicits a diseaseresponse.

“Promoter” refers to a DNA sequence capable of controlling theexpression of a coding sequence or functional RNA. The promoter sequenceconsists of proximal and more distal upstream elements, the latterelements often referred to as enhancers. Accordingly, an “enhancer” is aDNA sequence which can stimulate promoter activity and may be an innateelement of the promoter or a heterologous element inserted to enhancethe level or tissue-specificity of a promoter. Promoters may be derivedin their entirety from a native gene, or be composed of differentelements derived from different promoters found in nature, or evencomprise synthetic DNA segments. It is understood by those skilled inthe art that different promoters may direct the expression of a gene indifferent tissues or cell types, or at different stages of development,or in response to different environmental conditions. Promoters whichcause a gene to be expressed in most cell types at most times arecommonly referred to as “constitutive promoters”. New promoters ofvarious types useful in plant cells are constantly being discovered;numerous examples may be found in the compilation by Okamuro andGoldberg, 1989, Biochemistry of Plants 15:1-82. It is further recognizedthat since in most cases the exact boundaries of regulatory sequenceshave not been completely defined, DNA fragments of some variation mayhave identical promoter activity.

An “intron” is an intervening sequence in a gene that does not encode aportion of the protein sequence. Thus, such sequences are transcribedinto RNA but are then excised and are not translated. The term is alsoused for the excised RNA sequences. An “exon” is a portion of thesequence of a gene that is transcribed and is found in the maturemessenger RNA derived from the gene, but is not necessarily a part ofthe sequence that encodes the final gene product.

The “translation leader sequence” refers to a DNA sequence locatedbetween the promoter sequence of a gene and the coding sequence. Thetranslation leader sequence is present in the fully processed mRNAupstream of the translation start sequence. The translation leadersequence may affect processing of the primary transcript to mRNA, mRNAstability or translation efficiency. Examples of translation leadersequences have been described (Turner, R. and Foster, G. D., 1995, Mol.Biotechnol. 3:225).

The “3′ non-coding sequences” refer to DNA sequences located downstreamof a coding sequence and include polyadenylation recognition sequencesand other sequences encoding regulatory signals capable of affectingmRNA processing or gene expression. The polyadenylation signal isusually characterized by affecting the addition of polyadenylic acidtracts to the 3′ end of the mRNA precursor. The use of different 3′non-coding sequences is exemplified by Ingelbrecht et al., (1989) PlantCell 1:671-680.

“RNA transcript” refers to the product resulting from RNApolymerase-catalyzed transcription of a DNA sequence. When the RNAtranscript is a perfect complementary copy of the DNA sequence, it isreferred to as the primary transcript or it may be a RNA sequencederived from posttranscriptional processing of the primary transcriptand is referred to as the mature RNA. “Messenger RNA (mRNA)” refers tothe RNA that is without introns and that can be translated into proteinby the cell. “cDNA” refers to a DNA that is complementary to andsynthesized from a mRNA template using the enzyme reverse transcriptase.The cDNA can be single-stranded or converted into the double-strandedform using the klenow fragment of DNA polymerase I. “Sense” RNA refersto RNA transcript that includes the mRNA and so can be translated intoprotein within a cell or in vitro. “Antisense RNA” refers to a RNAtranscript that is complementary to all or part of a target primarytranscript or mRNA and that blocks the expression of a target gene (U.S.Pat. No. 5,107,065). The complementarity of an antisense RNA may be withany part of the specific gene transcript, i.e., at the 5′ non-codingsequence, 3′ non-coding sequence, introns, or the coding sequence.“Functional RNA” refers to antisense RNA, ribozyme RNA, or other RNAthat may not be translated but yet has an effect on cellular processes.

The term “operably linked” refers to the association of nucleic acidsequences on a single nucleic acid fragment so that the function of oneis affected by the other. For example, a promoter is operably linkedwith a coding sequence when it is capable of affecting the expression ofthat coding sequence (i.e., that the coding sequence is under thetranscriptional control of the promoter). Coding sequences can beoperably linked to at least one regulatory sequence in sense orantisense orientation.

The term “expression”, as used herein, refers to the production of afunctional end-product. Expression or overexpression of a gene involvestranscription of the gene and translation of the mRNA into a precursoror mature protein. “Antisense inhibition” refers to the production ofantisense RNA transcripts capable of suppressing the expression of thetarget protein. “Overexpression” refers to the production of a geneproduct in transgenic organisms that exceeds levels of production innormal or non-transformed organisms. “Co-suppression” refers to theproduction of sense RNA transcripts capable of suppressing theexpression of identical or substantially similar foreign or endogenousgenes (U.S. Pat. No. 5,231,020).

“Altered expression” refers to the production of gene product(s) intransgenic organisms in amounts or proportions that differ significantlyfrom that activity in comparable tissue (organ and of developmentaltype) from wild-type organisms.

“Mature” protein refers to a post-translationally processed polypeptide;i.e., one from which any pre- or propeptides present in the primarytranslation product have been removed. “Precursor” protein refers to theprimary product of translation of mRNA; i.e., with pre- and propeptidesstill present. Pre- and propeptides may be but are not limited tointracellular localization signals.

A “chloroplast transit peptide” is an amino acid sequence which istranslated in conjunction with a protein and directs the protein to thechloroplast or other plastid types present in the cell in which theprotein is made. “Chloroplast transit sequence” refers to a nucleotidesequence that encodes a chloroplast transit peptide. A “signal peptide”is an amino acid sequence which is translated in conjunction with aprotein and directs the protein to the secretory system (Chrispeels, J.J., (1991) Ann. Rev. Plant Phys. Plant Mol. Biol. 42:21-53). If theprotein is to be directed to a vacuole, a vacuolar targeting signal(supra) can further be added, or if to the endoplasmic reticulum, anendoplasmic reticulum retention signal (supra) may be added. If theprotein is to be directed to the nucleus, any signal peptide presentshould be removed and instead a nuclear localization signal included(Raikhel (1992) Plant Phys. 100:1627-1632).

“Transformation” refers to the transfer of a nucleic acid fragment intothe genome of a host organism, resulting in genetically stableinheritance. Host organisms containing the transformed nucleic acidfragments are referred to as “transgenic” organisms. The preferredmethod of cell transformation of rice, corn and other monocots is theuse of particle-accelerated or “gene gun” transformation technology(Klein et al., (1987) Nature (London) 327:70-73; U.S. Pat. No.4,945,050), or an Agrobacterium-mediated method using an appropriate Tiplasmid containing the transgene (Ishida Y. et al., 1996, NatureBiotech. 14:745-750).

Standard recombinant DNA and molecular cloning techniques used hereinare well known in the art and are described more fully in Sambrook, J.,Fritsch, E. F. and Maniatis, T. Molecular Cloning: A Laboratory Manual;Cold Spring Harbor Laboratory Press: Cold Spring Harbor, 1989(hereinafter “Sambrook”).

“PCR” or “Polymerase Chain Reaction” is a technique for the synthesis oflarge quantities of specific DNA segments, consists of a series ofrepetitive cycles (Perkin Elmer Cetus Instruments, Norwalk, Conn.).Typically, the double stranded DNA is heat denatured, the two primerscomplementary to the 3′ boundaries of the target segment are annealed atlow temperature and then extended at an intermediate temperature. Oneset of these three consecutive steps comprises a cycle.

An “expression construct” as used herein comprises any of the isolatednucleic acid fragments of the invention used either alone or incombination with each other as discussed herein and further may be usedin conjunction with a vector or a subfragment thereof. If a vector isused then the choice of vector is dependent upon the method that will beused to transform host plants as is well known to those skilled in theart. For example, a plasmid vector can be used. The skilled artisan iswell aware of the genetic elements that must be present on the vector inorder to successfully transform, select and propagate host cellscomprising any of the isolated nucleic acid fragments of the invention.The skilled artisan will also recognize that different independenttransformation events will result in different levels and patterns ofexpression (Jones et al., (1985) EMBO J. 4:2411-2418; De Almeida et al.,(1989) Mol. Gen. Genetics 218:78-86), and thus that multiple events mustbe screened in order to obtain lines displaying the desired expressionlevel and pattern. Such screening may be accomplished by Southernanalysis of DNA, Northern analysis of mRNA expression, Western analysisof protein expression, or phenotypic analysis. The terms “expressionconstruct” and “recombinant expression construct” are usedinterchangeably herein.

Cloned R-genes can be used to facilitate the construction of crop plantsthat are resistant to pathogens. In particular, transformationtechnology can be used to stack multiple single genes into an agronomicgermplasm without linked genomic sequences that accompany genestransferred by classical breeding techniques. Cloned R-genes also can beused to overcome the inability to transfer disease resistance genesbetween plant species by classical breeding.

The present invention concerns an isolated nucleic acid fragmentcomprising a nucleic acid sequence or subsequence thereof encoding analtered Pi-ta resistance polypeptide wherein the polypeptide has asingle amino acid alteration at position 918 which confers a resistancegene mediated defense response against a fungus comprising in its genomevirulent and/or avirulent AVR-Pita alleles. In another aspect, thepresent invention provides an isolated nucleic acid fragment that hasutility in controlling rice blast disease, caused by the fungusMagnaporthe grisea, in rice.

As discussed below, other subpopulations of M. grisea possess AVR-genesthat are homologous to that contained in strains that elicit the Pi-taspecific defense response in rice. Demonstrations that AVR-genes whichtrigger a Pi-ta-resistance gene-mediated defense response can be presentin M. grisea rice pathogens in subpopulation I, in Digitaria pathogensin subpopulation II, and in Pennisetum pathogens in subpopulation III,support broad utility for this gene in controlling M. grisea on therange of graminaceous hosts infected by this fungus.

Thus, it is believed that the isolated nucleic acid fragments of theinvention will have utility in controlling diseases caused by a funguscomprising in its genome virulent and/or avirulent AVR-Pita alleles. Forexample, this invention will have utility in controlling diseases causedby M. grisea on other cereal crops including, but not limited to, wheat,barley, corn, finger millet, sorghum, and pearl millet.

Alternatively, some virulent avr-pita alleles encode substantiallysimilar proteins (such as SEQ. ID NO:5 for G-198 and SEQ ID NO:13 forIna 72.2) to the avirulent AVR-Pita protein and yet fail to trigger aPi-ta-mediated resistance response. In its most extreme form, singleamino acid substitutions introduced either through spontaneousmutations, or by in vitro mutagenesis, eliminate the ability of AVR-Pitato trigger a Pi-ta-resistance gene-mediated defense response.

The genetic cross (cross 4360) that identified the PWL2 gene, anAVR-gene controlling host species specificity on weeping lovegrass(Sweigard et al., 1995, The Plant Cell 7:1221), also segregated for anadditional fungal gene that determined the ability of rice pathogens toinfect rice variety Yashiro-mochi. This second AVR-gene, AVR-Pita(formerly called AVR2-YAMO for Yashiro-mochi) was inherited from theparental strain, 4224-7-8, and was derived from the Chinese fieldisolate O-137 (collected in 1985 at the China National Rice ResearchInstitute in Hangzhou). In each of five complete tetrads derived fromcross 4360, four of eight ascospore progeny were able to infectYashiro-mochi and the others were not. Random spore analysis of cross4360 and subsequent crosses confirmed segregation of AVR-Pita. Avirulentprogeny from cross 4360 frequently produced a few fully pathogeniclesions on Yashiro-mochi. It was speculated that these rare lesionsmight be due to spontaneous mutations occurring at the AVR-Pita locus.Mutants that had lost function of AVR-Pita were isolated as described inSweigard et al (1995, The Plant Cell 7:1221). These mutants that werenow fully virulent toward Yashiro-mochi retained morphological andfertility characteristics as well as the MGR586 DNA fingerprintingprofiles of the presumptive parent. The host specificities of themutants toward rice varieties with other R-genes were unchanged.

Although dominance is not easy to assess for genes in predominantlyhaploid fungi like M. grisea, the occurrence of virulent mutantssuggested that the expressed form of this AVR gene functions to stopinfection of Yashiro-mochi, as predicted by the gene-for-genehypothesis. The genetic instability of the AVR-Pita gene aided in itscloning. The AVR-gene was found to cosegregate with a cluster ofphysical markers including the telomeric repeat sequence at the end of alinkage group in the M. grisea RFLP map produced from cross 4360(Sweigard et al., 1993, Genetic Maps, edited by S. J. O'Brien, ColdSpring Harbor Laboratory, pp 3.112-3.117). Spontaneous mutants that hadbecome virulent on Yashiro-mochi rice showed structural changes intelomeric restriction fragments that mapped with the avirulence gene,suggesting the gene resided within 1 to 2 kb of the tip of thechromosome (Valent and Chumley, 1994, The Rice Blast Disease, edited byZeigler, Leong and Teng, CAB International, Wallingford). Southernanalysis of genomic DNA from wild type avirulent strains and fromspontaneous mutants that had acquired deletions at the chromosome end,identified the sizes of the terminal chromosome fragment produced bydigestion of genomic DNA with various restriction enzymes. This analysissuggested that the AVR-gene resided within a telomeric 6.5 kb BglIIfragment that corresponded to the chromosome end. Cloning of thecorresponding telomeric fragment allowed demonstration that it didindeed contain the AVR-Pita gene, which functioned to transform virulentpathogens of rice cultivar Yashiro-mochi into avirulent strains onYashiro-mochi.

The AVR-Pita nucleic acid fragment isolated from the Chinese ricepathogen O-137 encodes a protein with 223 amino acids (SEQ ID NO:3).Amino acids 173-182 form a characteristic motif of a neutral zincmetalloproteinase and natural or in vitro mutation of the motif residuesdestroys AVR-gene activity, that is, it no longer transforms virulentstrains of the pathogen to avirulence on rice variety Yashiro-mochi(Valent and Chumley, 1994, The Rice Blast Disease, edited by Zeigler,Leong and Teng, CAB International, Wallingford). The predicted aminoacid sequence has low levels of homology to other metalloproteinasescharacterized from fungi (Genbank Accession numbers L37524 and S16547).The best characterized secreted fungal metalloprotease, NplI fromAspergillus oryzae, contains a 175 amino acid prepro-region thatprecedes a 177 amino acid mature region (Tatsumi et al., 1991, Mol. Gen.Genet. 228:97-103). The predicted AVR-Pita amino acid sequence exhibits35% homology and 29% identity with NplI, with the most significanthomology confined to the mature 177 amino acid form of NplI. Inaddition, alignment of the amino acid sequences of AVR-Pita and NplIshowed conservation of the cysteines involved in disulphide bonds in themature NplI protein. It was anticipated that the AVR-Pita isolatednucleic acid fragment encodes a preproprotein that is processed to amature metalloprotease containing 176 amino acids. Based on thisprediction, an AVR-Pita₁₇₆ expression construct was engineered toproduce directly the putative mature protease for functional analyses.

Functional AVR-Pita nucleic acid fragments have been cloned from M.grisea strains that infect host plants other than rice, and aredistantly related to rice pathogens in subpopulation Ia, including aDigitaria pathogen (JP34, also known as G-213, isolated in Japan) fromsubpopulation III, and a Pennisetum pathogen (BF17, isolated in BurkinaFaso) from subpopulation IV. The AVR-Pita nucleic acid fragment clonedfrom the Digitaria pathogen (SEQ ID NO:14) corresponds to a translatedamino acid sequence (SEQ ID NO:16) with 87.9% similarity and 84.7%identity to the O-137 AVR-Pita amino acid sequence when compared by theBestfit algorithm of the University of Wisconsin Computer group package9.1 (Devereux et al., 1984, Proc Natl Acad Sci USA 12:387-395). TheG-213 AVR-Pita (avirulence) nucleic acid fragment has the most divergentsequence identified which retains the ability to transform virulent ricepathogens into avirulent strains that elicit a Pi-ta resistance genemediated defense response. Conservation of AVR-gene function betweendistantly related M. grisea strains that infect different grass speciessuggests that a cloned Pi-ta resistance gene will be effective incontrolling the blast fungus on its other host plants, in addition torice. The present invention concerns an isolated nucleic acid fragmentcomprising a nucleic acid sequence or subsequence thereof encoding analtered Pi-ta resistance polypeptide wherein the polypeptide has asingle amino acid alteration at position 918 which confers a resistancegene mediated defense response against a fungus comprising in its genomevirulent and/or avirulent AVR-Pita alleles.

More specifically, it has been found that the single amino acidalteration at position 918 can be selected from the group consisting ofmethionine, cysteine, isoleucine, arginine, lysine, asparagine, leucine,and glutamine (M, C, I, R, K, N, L, and Q). As is shown in the examplesbelow, this single amino acid alteration in susceptible and resistantforms of the Pi-ta resistance protein correlate with recognitionspecificity.

Alteration of the amino acid at position 918 makes it possible togenerate mutant Pi-ta genes to recognize virulent AVR-Pita alleles forwhich no R-gene has currently been identified. Clearly, an ability to doso provides a valuable tool to control currently virulent strains of therice blast fungus and other pathogens.

In another aspect this invention concerns chimeric genes comprisingisolated nucleic acid fragments described herein operably linked to atleast one regulatory sequence. Also of interest are plants transformedwith such chimeric genes and seeds obtained from such plants.

Transgenic plants of the invention can be made using techniques wellknown to those of ordinary skill in the art, as is dicussed above, whichare capable of mounting a resistance gene mediated defense responseagainst a fungus comprising in its genome virulent and/or avirulentAVR-Pita alleles. Introduction of transgenes into plants, i.e.,transformation is well known to those skilled in the art. A preferredmethod of plant cell transformation is the use of particle-acceleratedor “gene gun” transformation technology (Klein et al. (1978) Nature(London) 327:70-73; U.S. Pat. No. 4,945,050). Examples of plants thatcan be transformed with such transgenes include, but are not limited to,monocots. Preferably, the monocot is a cereal. Most preferably, themonocot is rice, wheat, barley, corn, finger millet, sorghum, or pearlmillet.

In still another aspect, this invention concerns a method of conferringa resistance gene mediated defense response in plants against a funguscomprising in its genome virulent and/or avirulent AVR-Pita alleles inplants which comprises: (a) transforming a plant with a chimeric gene ofthe invention; and (b) selecting transformed plants of step (a) whichare resistant to a fungus comprising in it genome virulent and/oravirulent AVR-Pita alleles.

EXAMPLES

The present invention is further defined in the following Examples. Fromthe above discussion and these Examples, one skilled in the art canascertain the essential characteristics of this invention, and withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the invention to adapt it to various usages andconditions.

Unless otherwise stated, all parts and percentages are by weight anddegrees are Celsius. Techniques in molecular biology were typicallyperformed as described in Sambrook, J., Fritsch, E. F. and Maniatis, T.1989. Molecular cloning—A Laboratory Manual, 2^(nd) ed. Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y.

In some instances, amino acids are indicated by their one-letterdesignations widely used in the literature. For example A918 indicatesthat alanine (A) is at position 918 of the amino acid sequence of theprotein.

Example 1 Pathogen Strain Development for Identification of theCorresponding R-Gene

An avirulent isolate of the pathogen is necessary to identify a hostresistance gene, and the use of new pathogen strains may identifyresistance genes that have so far escaped detection. Two fungal strainsthat show virulence (that is, they fail to elicit an R-gene mediateddefense response) toward a rice variety with a particular R-gene areconsidered to lack the corresponding functional AVR-gene. However,although an avirulent strain of the fungus with a corresponding AVR-geneis necessary for identifying a host resistance gene, when two strainsare avirulent toward a particular rice variety, they could have the sameAVR-gene, or different AVR-genes triggering different R-gene mediateddefense responses. Use of new or uncharacterized fungal strains mayidentify previously unidentified R-genes. This caution is especiallyrequired when working with field isolate rice pathogens such as O-137,the source of the AVR-Pita gene. In addition, rice blast infectionassays are notoriously sensitive to environmental conditions, especiallyif the rice varieties have some level of general resistance to blast, orif the pathogens used are unstable in pathogenicity and morphologycharacteristics. Therefore, the use of thoroughly characterized pathogenstrains is key to success.

Multiple pathogen resources, including genetic populations segregatingfor the AVR-Pita gene, facilitated cloning the Pi-ta gene. Progenystrains such as 4360-R-17, 4360-R-27, 4360-R-30, and subsequentgeneration strain 4375-R-26 (Sweigard et al., 1995, The Plant Cell7:1221-1233) had improved characteristics for these studies. Because therice crosses in these experiments had at least two R-genes segregating,Pi-ta and Pi-km, and because pathogen cross 4360 was segregating forAVR-genes corresponding to both known R-genes, there was a need toobtain genetically characterized pathogen strains bearing singleAVR-genes. Such strains were needed to identify the effects ofindividual R-gene/AVR gene interactions on lesion development. There wasalso the need to maximize phenotypic differences between the resistantand susceptible interactions in order to assure accuracy of scoring theresistance phenotype among the rice progeny in the mapping population.Twenty-eight progeny strains were obtained from cross 4360 thatcontained AVR-Pita, but which did not contain AVR1-TSUY. It is believedthat AVR1-TSUY is the AVR-gene corresponding to the blast resistancegene Pi-km in the japonica variety Tsuyuake (Yamada et al. (1976) Ann.Phytopathol. Soc. Japan 42: 216-219). These 28 progeny were screened forstable ability to produce fully susceptible disease symptoms (Type4-Type 5 lesions) on one parental rice variety, and typical resistanceresponses (Type 0-Type 1 lesions) on the other rice variety (Valent etal., 1991, Genetics 127:87). M. grisea isolate 4360-R-62 was chosen as atest strain which contained only AVR-Pita. Out of an additional 9strains screened, 4360-R-67 was chosen as an excellent pathogen thatcontains only AVR1-TSUY.

In addition, mutants that have lost AVR-gene function are useful forcomparison to the parental strain from which they were derived. Forexample, strain CP987 (Sweigard et al. (1995) The Plant Cell7:1221-1233) was derived from strain 4360-R-17 by isolation of aspontaneous virulent mutant. The AVR-Pita gene in CP987 was inactivatedby a small deletion. Virulent mutants can easily be obtained fromavirulent field isolates or laboratory strains as described in Sweigardet al. (1995) The Plant Cell 7:1221-1233. In the opposite direction ofpathogen trait alteration, field isolate Guy11, which is virulent onYashiro-mochi (Yamada et al. (1976) Ann. Phytopathol. Soc. Japan 42:216-219), became avirulent on this host when it was transformed with acloned AVR-Pita gene to produce strain CP3285. Fungal transformation inthe rice blast system is routine, as described in Sweigard et al. (1995)Plant Cell 7:1221.

Example 2 Plant Infection and Evaluation

Infection assays were performed as previously described (Valent et al.,1991, Genetics 127:87). Conidia were collected from cultures grown onoatmeal agar plates by washing with a sterile 0.25% gelatin solution.Five to six individuals from each rice variety were sown in Metro-Mix®potting medium within plastic pots in a growth chamber. Plants weregrown on a day cycle of 14 hours of light, 28 degrees and 70 to 85%relative humidity. Night conditions were 22 degrees and 85% relativehumidity. Pots with two-week-old plants were placed into plastic bags inorder to maintain 95-100% relative humidity required for pathogenpenetration, and inoculated with pathogen strains such as but notlimited to 4360-R-62 or 4360-R-67 described in Example 1. A four mlaqueous suspension, containing 2.5×10⁵ conidia per ml, was sprayed ontothe plants using an artist's air brush (Pasha, size 1). The plastic bagscontaining the inoculated plants were then closed with a twist-tie, andwere incubated in low light conditions at room temperature for 24 hours.After 24 hours, the plants were removed from the bags, and were placedback in the growth chamber. Infection types, i.e., lesion symptoms, werescored 7 days after inoculation. A scale of 0-5 was used to classifyinfection phenotypes (Valent et al., 1991, Genetics 127:87-101). Lesiontypes of 0 and 1 were scored as resistant while lesion types 3 to 5 werescored as susceptible.

Example 3 Isolation of Nucleic Acid Fragments Encoding Avirulent andVirulent AVR-Pita Alleles

Isolation of AVR-Pita Genomic Nucleic Acid Fragments

The AVR-Pita genomic nucleic acid fragments from M. grisea strainsO-137, Ina 72, GUY1, G-198 and G-213 are all believed to contain 3introns within the AVR-Pita coding sequence. Southern blot analysisdetermined that strains O-137 and G-213 contain a single AVR-Pita genewhereas GUY11, G-198 and Ina 72 contain two AVR-Pita genes. Toefficiently express the AVR-Pita nucleic fragment in rice leaves underthe control of the CaMV 35S-Adh1 promoter, it was necessary to isolatethe cDNA. AVR-Pita nucleic acid fragments have been cloned and/orsequenced from M. grisea strains O-137, G-213, GUY11, G-198 and two fromIna 72, and it is possible to use any of them to prepare AVR-Pita₁₇₆nucleic acid fragments for the transient expression experiments.

Cloning of the AVR-Pita Gene from Strain O-137 by Cloning a TelomereFragment

AVR-Pita was mapped to one end of M. grisea linkage group 2c of theO-137-derived progeny strain 4224-7-8 (Sweigard et al., 1993. In GeneticMaps, 6^(th) edition, S. J. O'Brien, ed., Cold Spring Harbor LaboratoryPress, pp.3.112-3.117). In this analysis, the AVR-Pita geneco-segregated with the telomere repeat sequences at the end of thechromosome. Identification of telomeric DNA fragments was routinelyperformed. Standard protocols were used for restriction enzymedigestions, RNA and DNA gel blot analysis (Sambrook; Ausubel et al.,1994, Current Protocols in Molecular Biology, Greene PublishingAssociates/Wiley Interscience, New York) except as noted below forhybridizations with the oligonucleotide probe. The oligonucleotide[5′-(AACCCT)₄-3′] was synthesized and radiolabeled by kinase treatmentwith γ-³²P-ATP for detection of the hexameric telomere repeat sequenceby DNA gel blot analysis. Genomic DNAs were prepared as described(Sweigard et al., 1995, Plant Cell 7:1221-1233), digested withrestriction enzymes, electrophoresed on 0.7% agarose gels, and blottedto Hybond-N membranes. For hybridization with the telomeric repeatoligonucleotide, membranes were prehybridized in 6×SSPE, 5×Denhardts,0.5% SDS, 100 g/ml ssCT DNA for 2 hrs at 42° C., and hybridized withradiolabeled oligonucleotide overnight at 42° C. Membranes were washedfirst for 10 minutes in 2×SSPE, 0.1%SDS at room temperature and then for15 minutes in the same solution at 30-34° C.

Changes in the sizes of telomeric restriction fragments that correlatedwith a loss of avirulence toward Yashiro-mochi suggested the AVR-Pitagene is located directly adjacent to its linked telomere. Because cosmidlibraries were unlikely to contain chromosome ends, we decided to clonethis telomere specifically. Genomic DNA from the avirulent parentalstrain 4224-7-8 (Sweigard et al., 1995, The Plant Cell 7:1221-1233) wasfirst treated with BAL31 nuclease in order to remove any 3′ overhang ofthe G-rich strand and produce a blunt end at the telomere. The genomicDNA was treated with 0.125 units/ml of BAL31 nuclease (New EnglandBiolabs, Inc.) for 50 min at 30° C. as described (Richards and Ausubel(1988) Cell 53:127-136). Under these conditions, no visible decreaseoccurred in the size of the telomeric fragments as determined by DNA gelblot analysis. An enriched fraction of genomic DNA that would containthe telomere fragment was produced based on our deduction that the 6.5kb-BglII telomeric fragment that contains AVR-Pita does not contain anySalI sites. The Bal31 treated DNA was digested with the restrictionenzymes BglII and SalI and subjected to electrophoresis on a 0.8% lowmelting agarose gel, and used to create a genomic sub-library of 6- to7-kb fragments. DNA fragments in the size range of 7- to 8-kb wereeluted from the gel and ligated into the BamHI and EcoRV polylinkersites of pBluescript SK(+). The sub-library was screened fortelomere-containing clones using the telomeric oligonucleotide, andpositive clones were analyzed further. A single clone, designatedpCB780, was obtained with the predicted restriction fragments.

The AVR-Pita gene was identified in pCB780 by its ability to transformM. grisea virulent strains to avirulence towards rice containing Pi-ta.Fungal transformation for complementation analysis for function inconferring avirulence activity was performed as described (Sweigard etal., 1995, Plant Cell 7:1221-1233). To increase stability of the clonein M. grisea, pCB780 was cut with KpnI and partially digested withexonuclease III, resulting in plasmid pCB806 which had 123 bp of thetelomeric repeat (almost the entire repeat) deleted. pCB806 was then cutwith NcoI and XbaI to delete 4.5 kb of insert DNA, and the remaininginsert (1928 bp) and vector was blunted and re-ligated to create pCB813which was confirmed to contain an intact AVR-Pita gene using thefunctional assay described above. Nucleotide sequence of the insert inpCB813 (2 kb) was obtained, and used as basis of some of theoligonucleotide primers described herein (e.g., LF2C and LF2D).

Isolation of an AVR-Pita cDNA for the O-137 Allele AVR-Pita did notappear to be transcribed at detectable levels during axenic growth ofthe fungus in culture. We therefore subcloned the AVR-Pita genomiccoding sequence into a constitutive expression vector, pCB963, under thecontrol of the Aspergillus TrpC promoter and terminator (Staben et al.,1989). Strains transformed with pCB963 and grown in liquid cultureexpressed AVR-Pita as determined by RNA gel blot analysis using theDraIII—EcoRI fragment from pCB780 as a probe. A cDNA clone obtained fromthis transformed strain confirmed the positions of the three predictedintrons.

Specifically, the constitutive expression vector, pCB963, was producedas follows. The vector pCSN43 (Staben et al. (1989) Fungal GenetNewslett 36:79-81) was first modified to eliminate extra BamHI and ClaIsites by deletion of the smaller MluI-SacI fragment. The AVR-Pitagenomic coding sequence was cloned by PCR using oligonucleotides LF2C(SEQ ID NO:17) and LF2D (SEQ ID NO:18), designed to place a ClaI site(underlined in SEQ ID NO:17 below) at the start ATG and a BamHI site(underlined in SEQ ID NO:18 below) at the telomere end.

LF2C: 5′-GATCGAATCGATATGCTTTTTTATTCATTATTTTTTTTTC-3′ (SEQ ID NO:17)

LF2D: 5′-GATCGAGGATCCCCCTCTATTGTTAGATTGACC-3′ (SEQ ID NO:18)

The coding sequence of HPH in pCSN43 was then removed by digestion withClaI and BamHI and the ClaI/BamHI fragment containing the AVR-Pitagenomic coding sequence was inserted to produce pCB963.

Transgenic fungus containing pCB963 was grown in liquid culture forpurification of RNA. The Perkin Elmer Cetus—GeneAmp RNA PCR kit protocolwas used to reverse transcribe RNA using random hexamer priming followedby PCR amplification of cDNA using oligonucleotides LF2C (SEQ ID NO:17)and LF2D (SEQ ID NO:18). The cDNA fragment encoding AVR-Pita wasdigested with ClaI and BamHI and cloned into ClaI/BamHI-cut pBluescriptSK+ (Stratagene) to produce pCB979.

A 486 bp functional promoter fragment was amplified from the O-137genomic DNA clone pCB813 using primers LF2H (SEQ ID NO:19) and LF12 (SEQID NO:20).

LF2H: 5′-AAGCATATCGATAAAAATAATGTTAATTGTGCAG-3′ (SEQ ID NO:19)

LF12: 5′-GCCGAGTCGTTCTGAGGG-3′ (SEQ ID NO:20)

The 672 bp PCR product was end-filled using Klenow polymerase(Sambrook), digested with ClaI and cloned into the ClaI and HincII sitesof pCB979 to create pCB980.

Cloning of AVR-Pita Genomic Coding Sequences from Other Fungal Strains

Genomic coding sequences were amplified from genomic DNA isolated fromstrains G-198, Ina 72 and G-213 using primers LF2C (SEQ ID NO: 17), LF2D(SEQ ID NO:18), LF2C* (SEQ ID NO:21), GB84 (SEQ ID NO:22) and GB85 (SEQID NO:23) as shown in Table 3.

LF2C*: 5′-GATCGAATCGATATGCTTTTTTATTCATTGTTATTTTTATTTC-3′ (SEQ ID NO:21)

GB84: 5′-CCCTGGGATCCAACACTAACGTTATTTAACA-3′ (SEQ ID NO:22)

GB85: 5′-GCCGCATCGATATGCTTTTTTATTCATTTATATTTTA-3′(SEQ ID NO:23)

In each case, the 960 bp PCR product obtained was digested with BamHIand ClaI. pCB980 was also digested with BamHI and ClaI, and the 3394 bpfragment containing both the functional AVR-Pita promoter from strainO-137 and pBluescript SK+ vector sequences was gel-purified. Thedigested 945 bp PCR product was then cloned into the 3394 bp vectorfragment. This cloning step essentially replaced the 725 bp O-137AVR-Pita cDNA fragment with each of the genomic AVR-Pita nucleic acidfragments from G-198, Ina72 and G-213.

TABLE 3 Construction of Plasmids Containing AVR-Pita Genomic FragmentsFrom Various M. grisea Strains Resulting AVR-Pita Allele PhenotypePrimers used for PCR Plasmid G-198 Virulence LF2C (SEQ ID NO:17),pCB1447 LF2D (SEQ ID NO:18) Ina72.1 Avirulence LF2D (SEQ ID NO:18),pCB2076 LF2C* (SEQ ID NO:21) Ina72.2 Virulence LF2D (SEQ ID NO:18),pCB2077 LF2C* (SEQ ID NO:21) G-213 Avirulence GB84 (SEQ ID NO:22),pCB1965 GB85 (SEQ ID NO:23)

All genomic AVR-Pita nucleic acid fragments isolated thus far arebelieved to contain the three intron sequences. Predicted splicing ofthe introns was again confirmed by isolation of a cDNA clone encodingAVR-Pita of G-198 as described below. The cDNA sequence for the G-198allele is set forth in SEQ ID NO:4. For all other AVR-Pita clones, thethree intron sequences may be removed to generate a cDNA clone, usingthe appropriate oligonucleotides, subcloning and/or PCR techniques wellknown to those skilled in the art. The AVR-Pita genomic sequences fromIna 72 are set forth in SEQ ID NO:8 (Ina 72.1) and SEQ ID NO:11 (Ina72.2), and corresponding predicted cDNA sequences derived from thesegenomic sequences are respectively set forth in SEQ ID NO:9 and SEQ IDNO:12. The genomic sequence of the AVR-Pita nucleic acid fragment fromG-213 is set forth in SEQ ID NO:14, and a predicted cDNA sequence ofAVR-Pita from G-213 derived from SEQ ID NO:14 is set forth in SEQ IDNO:15.

To obtain the sequence of a virulent allele from GUY11, an AVR-Pitanucleic acid fragment was amplified from genomic GUY11 DNA by PCR usingprimers LF2D (SEQ ID NO:18) and LF2C (SEQ ID NO:17). The PCR product wassequenced directly after purification using a QIAquick™ PCR PurificationKit (Qiagen) The genomic sequence thus obtained is set forth in SEQ IDNO:59.

FIG. 1 is an alignment of the AVR-Pita amino acid sequences derived fromnucleotide sequences obtained from M. grisea strains O-137, G-213, Ina72, G-198, and GUY11.

Isolation of AVR-Pita cDNA Nucleic Acid Fragments for Expression of thePutative Mature Protease in Plants

Infection assays using pathogen strains O-137 and G-198 were performedas previously described (Valent et al., 1991, Genetics 127:87) and asrecited in Example 2.

pCB980 (SEQ ID NO:1) contains O-137 AVR-Pita cDNA, which was clone dusing the method desribed above.

Plasmid pAVR3 contains nucleotides 139-672 of the AVR-Pita nucleic acidfragment (SEQ ID NO:2) from M. grisea strain O-137 encoding thepredicted mature protease plus one additional N-terminal amino acid(AVR-Pita₁₇₇, beginning with Ile-47 of the preproprotein) and a startcodon met fused to the 35S/Adh1-6 promoter in vector pML 142. TheAVR-Pita nucleic acid fragment was amplified by PCR from AVR-Pita cDNAusing primers AV1 (SEQ ID NO:24) an d AV3 (SEQ ID NO:25), digested withPmlI and KpnI, blunted with Klenow polymerase and cloned into pML142that had also been cut with PmlI and KpnI and blunted with Klenowpolymerase, resulting in pAVR3.

AV1: 5′-GCCGGCACGTGCCATGATTGAACGCTATTCCCAATG-3′ (SEQ ID NO:24)

AV3: 5′-GCCGGGATCCCCCTCTATTGTTAGATTGAC-3′ (SEQ ID NO:25)

The coding sequence for the predicted mature protease (beg inning withGlu-48 of the preproprotein) was obtained by PCR-amplification from theAVR-Pita nucleic acid fragment (SEQ ID NO:2) in plasmid pAVR3 usingoligonucleotides YL30 containing an in-frame NcoI site (SEQ ID NO:26)and YL37 (SEQ ID NO:27).

YL30: 5′-ACAACAAGCCGGCACGTGCCATGGAACGCT-3′ (SEQ ID NO:26)

YL37: 5′-TCCTTCTTTAGGTACCGCTCTCTC-3′ (SEQ ID NO:27)

The PCR fragment was cloned NcoI/KpnI into pML142 resulting in vectorpCB1947. This was done to eliminate the Ile-47 codon (aft) in pAVR3 andgenerate AVR-Pita₁₇₆. Additional details are described in PCTPublication No. WO 00/08162, the disclosure of which is herebyincorporated by reference.

To isolate AVR-Pita cDNA nucleic acid fragments from strain G-198,plasmid pCB1447 was transformed into M. grisea strain CP987 andtransformants containing this plasmid were used to infect susceptibleTsuyuake rice. Nucleic acid fragments comprising the AVR-Pita cDNAcoding sequence from strain G-198 was amplified by RT-PCR from mRNAisolated from infected rice leaf tissue 72 hours after inoculation with2×10⁶ spores ml⁻¹ of M. grisea strain CP3402 (CP987 transformed withpCB1447) using primers GB183 (SEQ ID NO:28) and GB184 (SEQ ID NO:29)containing NcoI and Kpn sites, respectively.

GB183: 5′-GGGCTTCCATGGAACGCTATTCCCAATGTTCAG-3′ (SEQ ID NO:28)

GB184: 5′-CACTAAGGTACCTTAACATATTTATAACGTGCAC-3′ (SEQ ID NO:29)

The PCR product was digested with NcoI and KpnI and this was cloned intopML142 (described in WO 00/08162). pML 142 had also been digested withNcoI and KpnI and the 5.1 kb fragment isolated and purified from anagarose gel. The digested G-198 PCR nucleic acid fragment was ligatedwith the 5.1 kb pML 142 nucleic acid fragment using techniques familiarto those skilled in the art to generate plasmid pCB2148.

Example 4 Mutagenesis of Pi-ta at Position 918 to Create a Library ofPi-ta Plasmids With all 20 Amino Acids Represented at Position 918

Mutating the Pi-ta gene at codon 918 to create a set of modified Pi-tagenes with each of the 20 amino acids represented at position 918required a two step process which is described below. Two amino acidswere already represented, A918 (alanine at position 918) occurs inresistant forms of Pi-ta while S918 (serine at position 918) occurs insusceptible forms.

A full-length Pi-ta cDNA fragment was cloned using reverse transcriptase(RT) PCR and subcloning. The approach involved isolating mRNA fromtransgenic Nipponbare line 27-4-8-1 which contained a transgenecomprising a genomic Pi-ta nucleic acid fragment from Oryza sativavariety Yashiro-mochi operably linked to the CaMV 35S promoter(described in WO 00/01862, Example 10, expression construct 3) and whichwas shown on a northern blot to overexpress Pi-ta. First strand cDNA wassynthesized using the isolated mRNA fraction as template and theoligonucleotide GB67 (SEQ ID NO:30) as primer.

GB67:5′-CCATTAAGCTTGGTTTCAAACAATC-3′ (SEQ ID NO:30).

A partial Pi-ta cDNA (2.1 kb) was amplified from first strand cDNA usingprimers F12-1 (SEQ ID NO:31) and GB67 (SEQ ID NO:30).

F12-1: 5′-GTGGCTTCCATTGTTGGATC-3′ (SEQ ID NO:31)

It was then cloned into pSL1180 (Pharmacia) using the BamHI (restrictionsite present in the Pi-ta nucleic acid fragment) and HindIII(restriction site present in GB67 sequence) cloning sites. To obtain afull-length synthetic cDNA, a 706 bp NcoI-BamHI fragment containing the5′ end of the Pi-ta coding sequence was isolated from pCB1649 (describedin WO 00/08162) and cloned into the NcoI-BamHI site upstream of the 2090bp BamHI-HindIII partial Pi-ta cDNA fragment to create a full-lengthpromoter-less Pi-ta cDNA. DNA sequence analysis determined that therewas a 2 bp deletion present at codon 796 (probably a PCR artifact)resulting in a frameshift mutation that would have truncated thepredicted Pi-ta protein by 119 amino acids. This was corrected byreplacing a 1400 bp SphI-BglII fragment with the corresponding fragmentfrom pCB1649 which contained the correct sequence, to create pCB1906.DNA sequence analysis also determined that the predicted intron wasprecisely spliced in this synthetic cDNA. A native Pi-ta promoterfragment (2425 bp) was added by cloning a 3173 bp EcoRI fragment frompCB1649 into the EcoRI sites of pCB1906, resulting in plasmid pCB1926which contained the final Pi-ta cDNA construct (FIG. 2C) comprising 2425bp of the native Pi-ta promoter (nucleotides 1 to 2425 in SEQ ID NO:57)and Pi-ta cDNA (nucleotides 2426-5212 in SEQ ID NO:57) from rice varietyYashiro-mochi. The deduced amino acid sequence of the protein encoded bythis particular Pi-ta cDNA is set forth in SEQ ID NO:58.

Plasmid pCB2020 was produced by amplifying a susceptible Pi-ta nucleicacid fragment from susceptible C101A51 rice (Mackill and Bonman (1992)Phytopathology 82:746-749) genomic DNA in a PCR reaction using primersGB61 (SEQ ID NO:32) and GB67 (SEQ ID NO:30).

GB61: 5′-CAATGCCGAGTGTGCAAAGA-3′ (SEQ ID NO:32)

The resulting 440 bp PCR fragment was digested with BglII and HindIIIand cloned into plasmid pCB1926 that had been digested with the sameenzymes (and the corresponding 4900 bp nucleic acid fragment isolatedand purified from an agarose gel). This cloning step produced plasmidpCB2020 which is identical to pCB1926 except that codon 918 encodes Sand not A.

The remaining 18 amino acid modifications were introduced into Pi-ta byfirst amplifying the fragment from plasmid pCB1926 using PCR primer GB60(SEQ ID NO:33) and a set of 18 primers GB164-GB179, GB181 and GB182 (SEQID NOS:34-51) in 18 separate PCR reactions, one for each primer.

GB60: 5′-CAATGCCGAGTGTGCAAAGG-3 (SEQ ID NO:33)

GB164: 5′-AGGCGAGTCGACGTTTCAAACAATCATCAAGTCAGGTTGAAGATGCATCTCAGGTAAAGATAGAAGC-3′ (SEQ ID NO:34)

GB165: 5′-AGGCGAGTCGACGTTTCAAACAATCATCAAGTCAGGTTGAAGATGCATATCAGGTAAAGATAGAAGC-3′ (SEQ ID NO:35)

GB166: 5′-AGGCGAGTCGACGTTTCAAACAATCATCAAGTCAGGTTGAAGATGCATCCTAGGTAAAGATAGAAGC-3′ (SEQ ID NO:36)

GB167: 5′-AGGCGAGTCGACGTTTCAAACAATCATCAAGTCAGGTTGAAGATGCATCTTAGGTAAAGATAGAAGC-3′ (SEQ ID NO:37)

GB168: 5′-AGGCGAGTCGACGTTTCAAACAATCATCAAGTCAGGTTGAAGATGCATATTAGGTAAAGATAGAAGC-3′ (SEQ ID NO:38)

GB169: 5′-AGGCGAGTCGACGTTTCAAACAATCATCAAGTCAGGTTGAAGATGCATCATAGGTAAAGATAGAAGC-3′ (SEQ ID NO:39)

GB170: 5′-AGGCGAGTCGACGTTTCAAACAATCATCAAGTCAGGTTGAAGATGCATAATAGGTAAAGATAGAAGC-3′ (SEQ ID NO:40)

GB171: 5′-AGGCGAGTCGACGTTTCAAACAATCATCAAGTCAGGTTGAAGATGCATTGTAGGTAAAGATAGAAGC-3′ (SEQ ID NO:41)

GB172: 5′-AGGCGAGTCGACGTTTCAAACAATCATCAAGTCAGGTTGAAGATGCATCCAAGGTAAAGATAGAAGC-3′ (SEQ ID NO:42)

GB173: 5′-AGGCGAGTCGACGTTTCAAACAATCATCAAGTCAGGTTGAAGATGCATACAAGGTAAAGATAGAAGC-3′ (SEQ ID NO:43)

GB174: 5′-AGGCGAGTCGACGTTTCAAACAATCATCAAGTCAGGTTGAAGATGCATGTAAGGTAAAGATAGAAGC-3′ (SEQ ID NO:44)

GB175: 5′-AGGCGAGTCGACGTTTCAAACAATCATCAAGTCAGGTTGAAGATGCATGAAAGGTAAAGATAGAAGC-3′ (SEQ ID NO:45)

GB176: 5′-AGGCGAGTCGACGTTTCAAACAATCATCAAGTCAGGTTGAAGATGCATTTGAGGTAAAGATAGAAGC-3′ (SEQ ID NO:46)

GB177: 5′-AGGCGAGTCGACGTTTCAAACAATCATCAAGTCAGGTTGAAGATGCATGTGAGGTAAAGATAGAAGC-3′ (SEQ ID NO:47)

GB178: 5′-AGGCGAGTCGACGTTTCAAACAATCATCAAGTCAGGTTGAAGATGCATCAGAGGTAAAGATAGAAGC-3′ (SEQ ID NO:48)

GB179: 5′-AGGCGAGTCGACGTTTCAAACAATCATCAAGTCAGGTTGAAGATGCATCGGAGGTAAAGATAGAAGC-3′ (SEQ ID NO:49)

GB181: 5′-AGGCGAGTCGACGTTTCAAACAATCATCAAGTCAGGTTGAAGATGCATGCCAGGTAAAGATAGAAGC-3′ (SEQ ID NO:50)

GB182: 5′-AGGCGAGTCGACGTTTCAAACAATCATCAAGTCAGGTTGAAGATGCATAACAGGTAAAGATAGAAGC-3′ (SEQ ID NO:51)

The resulting 440 bp PCR nucleic acid fragments were digested with BglIIand SalI and the 322 bp fragment ligated into plasmid pCB2114 (describedbelow) that had been digested with the same enzymes. This replaced theresistant Pi-ta 322 bp BglII-SalI fragment in plasmid pCB2114 with anucleic acid fragment containing the altered codon at position 918. Thiscloning step produced plasmids pCB2153-pCB2170 (Table 4). PlasmidpCB2114 comprises nucleotide sequence encoding the Pi-ta leucine richdomain (LRD) described in WO 00/01862 which was amplified in a PCRreaction with primers GB47 (SEQ ID NO:52) and GB107 (SEQ ID NO:53). The1050 bp PCR fragment was digested with EcoRI and SalI and cloned intoplasmid pGAL4-BD-Cam (Stratagene) that had been digested with the sameenzymes.

GB47: 5′-AATGCAGAATTCACAACACCACTAGCAGGTTTG3′ (SEQ ID NO:52)

GB107: 5-AGGCGAGTCGACGTTTCAAACAATCATCAAGTCAGG-3′ (SEQ ID NO:53)

The second cloning step required the modified Pi-ta fragment in plasmidspCB2153-pCB2170 to be amplified with primers GB60 (SEQ ID NO:33) andGB67 (SEQ ID NO:30) in 18 separate PCR reactions. GB67 (SEQ ID NO:30)adds a HindIII cloning site after the TGA stop codon. The resulting 18PCR fragments were digested with BglII and HindIII and cloned intoplasmid pCB1926 that had been digested with the same enzymes (and thecorresponding 4900 bp nucleic acid fragment isolated and purified froman agarose gel). This cloning step generated plasmids pCB2171-pCB2188(Table 4). Table 4 identifies the plasmids with respect to the aminoacid at position 918 of the Pi-ta protein encoded by the nucleic acidfragments contained in the plasmids. For example, the table indicatesthat pCB2153 (line 3) contains a nucleic acid fragment encoding a Pi-taprotein in which the amino acid at position 918 is a glutamic acidresidue (E) instead of an alanine residue (A); this change was broughtabout by changing the Pi-ta nucleotide sequence via PCR as describedabove using oligonucleotides SEQ ID NO:33 and SEQ ID NO:34 (column 2).Plasmid pCB2153 was then used to make pCB2171 (column 4) as describedabove; pCB2171 was the one actually used (“transient assay plasmid”) inthe transient assay experiments described in Example 5. The sequence ofeach of these plasmids was verified by sequencing using primers that areenclosed in Example 7 in WO 00/01862. This resulting set of plasmidspCB2171-pCB2188, pCB1926 and pCB2020 represented a Pi-ta nucleic acidfragment with all 20 possible amino acid combinations at position 918linked to a native 2424 bp Pi-ta promoter nucleic acid fragment.

TABLE 4 Construction of Nucleic Acid Fragments Encoding Pi-ta ProteinsWith All Possible 20 Amino Acids Represented at Position 918 of thePi-ta Protein Oligonucleotide used with SEQ ID NO:33 in PCR to introduceamino Amino Acid Plasmid acid alteration Alteration Use pCB1926 — Pi-taTransient assay plasmid (resistant)A₉₁₈ pCB2020 — Pi-ta Transient assayplasmid (susceptible) S₉₁₈ pCB2153 SEQ ID NO:34 A₉₁₈-E Progenitor topCB2171 pCB2154 SEQ ID NO:35 A₉₁₈-D Progenitor to pCB2172 pCB2155 SEQ IDNO:36 A₉₁₈-R Progenitor to pCB2173 pCB2156 SEQ ID NO:37 A₉₁₈-KProgenitor to pCB2174 pCB2157 SEQ ID NO:38 A₉₁₈-N Progenitor to pCB2175pCB2158 SEQ ID NO:39 A₉₁₈-M Progenitor to pCB2176 pCB2159 SEQ ID NO:40A₉₁₈-I Progenitor to pCB2177 pCB2160 SEQ ID NO:41 A₉₁₈-T Progenitor topCB2178 pCB2161 SEQ ID NO:42 A₉₁₈-W Progenitor to pCB2179 pCB2162 SEQ IDNO:43 A₉₁₈-C Progenitor to pCB2180 pCB2163 SEQ ID NO:44 A₉₁₈-YProgenitor to pCB2181 pCB2164 SEQ ID NO:45 A₉₁₈-F Progenitor to pCB2182pCB2165 SEQ ID NO:46 A₉₁₈-Q Progenitor to pCB2183 pCB2166 SEQ ID NO:47A₉₁₈-H Progenitor to pCB2184 pCB2167 SEQ ID NO:48 A₉₁₈-L Progenitor topCB2185 pCB2168 SEQ ID NO:49 A₉₁₈-P Progenitor to pCB2186 pCB2169 SEQ IDNO:51 A₉₁₈-V Progenitor to pCB2187 pCB2170 SEQ ID NO:50 A₉₁₈-GProgenitor to pCB2188 pCB2171 SEQ ID NO:34 A₉₁₈-E Transient assayplasmid pCB2172 SEQ ID NO:35 A₉₁₈-D Transient assay plasmid pCB2173 SEQID NO:36 A₉₁₈-R Transient assay plasmid pCB2174 SEQ ID NO:37 A₉₁₈-KTransient assay plasmid pCB2175 SEQ ID NO:38 A₉₁₈-N Transient assayplasmid pCB2176 SEQ ID NO:39 A₉₁₈-M Transient assay plasmid pCB2177 SEQID NO:40 A₉₁₈-I Transient assay plasmid pCB2178 SEQ ID NO:41 A₉₁₈-TTransient assay plasmid pCB2179 SEQ ID NO:42 A₉₁₈-W Transient assayplasmid pCB2180 SEQ ID NO:43 A₉₁₈-C Transient assay plasmid pCB2181 SEQID NO:44 A₉₁₈-Y Transient assay plasmid pCB2182 SEQ ID NO:45 A₉₁₈-FTransient assay plasmid pCB2183 SEQ ID NO:46 A₉₁₈-Q Transient assayplasmid pCB2184 SEQ ID NO:47 A₉₁₈-H Transient assay plasmid pCB2185 SEQID NO:48 A₉₁₈-L Transient assay plasmid pCB2186 SEQ ID NO:49 A₉₁₈-PTransient assay plasmid pCB2187 SEQ ID NO:51 A₉₁₈-V Transient assayplasmid pCB2188 SEQ ID NO:50 A₉₁₈-G Transient assay plasmid

Example 5 Transient Particle Bombardment Assay for Testing Efficacy ofTriggering Pi-ta Mediated Resistance by Modified Pi-ta Genes Toward theVirulent AVR-Pita Allele from M. grisea Strain G-198

Particle Bombardment Demonstrates that AVR-Pita from avirulent M. griseaStrain O-137 But Not avr-pita from virulent M. grisea Strain G-198Elicits a Hypersensitive Response in Resistant Rice containing Pi-ta

As is standard practice to those skilled in the art, high velocitybiolistic bombardment of plant tissue with particles coated withrecombinant expression constructs of interest results in transientexpression of the nucleic acid fragments from the introduced plasmids.Function of disease resistance genes can be demonstrated in transientleaf bombardment experiments using reporter gene expression to assay fortriggering of the hypersensitive cell death resistance response. Such anassay has demonstrated utility for analyzing function of the Pi-taresistance gene and is described in WO 00/08162.

In order to overcome the obstacle to uniform incorporation of the fungalAVR-gene within the plant tissue, introduction of an AVR-Pita expressionconstruct was tested by co-bombardment along with the GUS reporter gene.In particular, constructs were engineered that express the putativemature protease (AVR-Pita₁₇₆) from strains O-137 (pCB1947) and G-198(pCB2148) under control of the 35S promoter for constitutive expressionin plant cells.

To determine if co-expression of the AVR-Pita₁₇₆ construct from strainsO-137 and G-198 triggers Pi-ta mediated defense responses whenintroduced into Pi-ta-containing plant cells, seedlings fromPi-ta-plants (Yashiro-mochi and YT14) and plants that lack Pi-ta(Nipponbare and YT16) were co-bombarded with pCB1947 or pCB2148containing the 35S/Adh1-6::AVR-Pita₁₇₆ gene construct and pML63 (whichis described in WO 00/08162) containing the 35S::GUS reporter gene. YT14and YT16 are described in WO 00/08162. Nipponbare is a rice variety thatis widely studied (e.g., Mao et al. (2000) Genome Res 10:982-990). Seedswere germinated on leaf assay media: {fraction (1/2 )} strength MSmedium (Murashige and Skoog, 1962, Physiol. Plant. 15:473-497)supplemented with 100 mg casein hydrolysate and 0.5% agarose for a weekin an incubator at 25° C. for 48 hours in 12 hr photoperiod with a 100μEm⁻²s⁻¹ of cool, white light. Two-leaf seedlings were excised from theagar medium using a surgical razor and placed in a petri dish containinga prewetted filter paper. Plantlets were labeled at the base with apermanent marker for identification. Biolistic bombardment of theseedlings was performed using Bio-Rad PDS-1000/He apparatus and 1150-psirupture disks. Gold particles (0.6 μm diameter) were prepared accordingto the instructions provided by the manufacturer. For eachcobombardment, 1 μg of gold particles was coated with 1.5 μg of 35S/GUSand 1 μg of other plasmids. After bombardment, seedlings were maintainedat 25° C. for 48 hours in Petri dishes containing prewetted filterpaper. Leaves were cleared in 70% ethanol and histochemically assayedfor β-Glucuronidase (GUS) activity using 5-bromo-4-chloro-3-indolylglucuronide (X-gluc) as a substrate (Jefferson, 1987, Plant Mol. Bio.Rep. 5:387-405).

The AVR-Pita₁₇₆ expression construct pCB1947 mediated a strikingdecrease in GUS expression (suggesting cell death prior to expression),when cobombarded with the GUS construct into YT14 leaves containing theendogenous Pi-ta gene. This effect was not observed in susceptible YT16leaves treated the same way. A virulent form of AVR-Pita₁₇₆ in constructpCB2148 failed to decrease GUS expression in either resistant TY14 orsusceptible YT16 leaves. Thus, only expression of the AVR-Pita codingsequence from O-137 triggers the rice defense response when introduceddirectly into rice leaves containing the endogenous Pi-ta gene.

In the second part of this experiment, a third construct was added,pCB1926. Seedlings from Pi-ta-plants (Yashiro-mochi and YT14) and plantsthat lack Pi-ta (Nipponbare and YT16) were co-bombarded with the35S/Adh1-6::AVR-Pita₁₇₆ gene construct pCB1947 or pCB2148, the Pi-tacDNA under the control of its native promoter (pCB1926) and the 35S::GUSreporter gene. In this case susceptible YT16 rice leaves responded inthe same way as resistant YT14 rice leaves to the avirulent AVR-Pita₁₇₆construct from strain O-137. It is therefore possible to add Pi-taexogenously to susceptible rice leaves to elicit a HR response toAVR-Pita. No HR response was observed towards the virulent avr-Pitaconstruct from virulent strain G-198.

This assay forms the basis for the next experiment described where 18new Pi-ta variants described in Example 4 above were then tested fortheir ability to encode a novel Pi-ta protein that responds to virulentavr-Pita proteins to elicit an HR.

Testing Pi-ta Transient Expression Vectors Encoding Pi-ta ProteinsAltered at Position 918

The 20 vectors pCB1926, pCB2020 and pCB2171-pCB2188 described in Example4 above were tested in the transient assay using the conditionsdescribed above. All were co-bombarded with pML63 which contains a35S::GUS reporter gene and either pCB1947 or pCB2148 which containdifferent 35S/Adh1-6::AVR-Pita₁₇₆ gene constructs. Results aresummarized in Table 5. In the table, “−” indicates lack of GUS stainingthat is indicative of the hypersensitive response (there is productiverecognition between the AVR-Pita and Pi-ta gene products); “+/−”indicates a few rare GUS loci that may indicate productive recognitionbetween the AVR-Pita and Pi-ta gene products; “+” indicates a smallnumber of GUS loci indicative of possible albeit suboptimal recognitionbetween the AVR-Pita and Pi-ta gene products; and “++” and “+++”indicate multiple GUS loci indicative of no productive recognitionbetween the AVR-Pita and Pi-ta gene products. Plates 1 and 2 representtwo replicates for each AVR-Pita/Pi-ta combination, with each platehaving two test seedlings. “Repeats” indicates the number of times aparticular experiment was repeated.

TABLE 5 Effect of Amino Acid at Position 918 on Ability of Pi-ta toRecognize AVR-Pita from M. grisea strain O-137 and avr-pita from M.grisea strain G-198 GUS Activity @ 48 hr AVR-Pita avr-pita (O-137)(G-198) (pCB1947) (pCB2148) No AVR gene Pi-ta Plate Plate Plate PlatePlate Plate Constructs 1 2 1 2 1 2 Repeats Conclusion A₉₁₈ − − + ++ 4resistant control - recognizes (Resistant) avirulent S₉₁₈ ++ ++ + ++ 4susceptible control - no (Susceptible) recognition G₉₁₈ ++ +++ ++ ++ 1not functional - no recognition V₉₁₈ ++ ++ + ++ 1 not functional - norecognition E₉₁₈ − + − ++ 1 not functional - no recognition D₉₁₈ ++++++ + + 1 not functional - no recognition R₉₁₈ − − − + 1 recognizesavirulent-reduced recognition? K₉₁₈ − + − − 1 recognizesvirulent-reduced recognition? N₉₁₈ − + − − 1 recognizes virulent-reducedrecognition? M₉₁₈ − − − − ++ ++ 3 recognizes both avirulent and virulentI₉₁₈ ++ ++ − − 3 recognizes virulent - switched specificity H₉₁₈ + + + +1 not functional - no recognition L₉₁₈ +/− +/− +/− +/− 1 recognizes bothavirulent and virulent P₉₁₈ + + + ++ 1 not functional - no recognitionT₉₁₈ + +/− + +/− 1 not functional - no recognition W₉₁₈ − +/− − + 1 C₉₁₈+/− − − − 2 recognizes both avirulent and virulent Y₉₁₈ ++ + + + 1 notfunctional - no recognition F₉₁₈ + + − + 1 Q₉₁₈ + + − − 1 recognizesavirulent-reduced recognition?

Consistent with disease phenotypes observed when Yashiro-mochi ischallenged with various M. grisea strains, Table 5 indicates that thePi-ta protein from Yashiro-mochi (A918) recognizes the AVR-Pita proteinfrom O-137 (which in planta results in disease resistance phenotype whenYashiro-mochi is challenged with O-137) but not the AVR-Pita form fromG-198 (which in planta results in disease susceptibility phenotype whenYashiro-mochi is challenged with G-198). In addition, the resultsdescribed in Table 5 identified two Pi-ta constructs encoding Pi-taproteins with amino acid 918 changed to either M or C that recognizedboth avirulent (AVR-Pita from O-137) and virulent (avr-pita from G-198)AVR-Pita gene products. As can be seen from the table, M918 Pi-ta gaverise to multiple GUS loci when there was no AVR-Pita gene co-bombardedwith the other constructs, indicating that by itself, M918 is not ableto cause the hypersensitive response; this indicated that the absence ofGUS loci observed when AVR-Pita gene was present was indeed due toproductive recognition between AVR-Pita and Pi-ta gene products and notfrom other reasons like autoactivation of the Pi-ta protein leading tothe hypersensitive response. Another form with an I at position 918 hadswitched specificity relative to the Pi-ta protein from Yashiro-mochi(A918) since it only recognized the virulent G-198 allele but not theO-137 allele. Other constructs encoding proteins containing R, K, N, L,and Q at position 918 also resulted in recognition of the G-198 allele.

These data suggest that it is possible to alter the range of AVR-Pitaalleles that Pi-ta can recognize by changing the amino acid at position918 of the Pi-ta protein. Consequently, transgenic rice plants that areable to recognize M. grisea strain G-198 and/or other strains containingan AVR-Pita gene substantially similar to that found in SEQ ID NO:4 andundergo an HR or disease resistance response may be obtained bytransforming rice with a modified Pi-ta nucleic acid fragment whichencodes a Pi-ta protein that has at position 918 an amino acid selectedfrom the grout, consisting of M, C, I, R, K, N, L and Q.

Additionally, modified Pi-ta nucleic acid fragments which encode a Pi-taprotein that has at position 918 an amino acid selected from the groupconsisting of M, C, I, R, K, N, L and Q, may be introduced into ricevarieties that already have a functional Pi-ta (A918), resulting inlines which can mount a disease resistance response to a broader rangeof M. grisea strains.

Example 6 Construction of Chimeric Genes Encoding Modified Pi-taProteins for Stable Rice Transformation

Plasmids comprising nucleic acid fragments encoding the altered forms ofPi-ta identified in Example 5 were constructed using vector pCB1926 andthus do not have a terminator sequence. For stable rice transformation,the vector pCB2022 is the preferred Pi-ta format for a Pi-ta nucleicacid fragment as in addition to the 2425 bp of Pi-ta native promotersequence and 2784 bp of Pi-ta coding sequence it contains an In2-1terminator sequence (SEQ ID NO:60). Plasmid pCB2022 has been depositedwith the ATCC. To make the precursor plasmid pCB2021, the entire 5222 bpEcoRI-HindIII Pi-ta nucleic acid fragment was excised from plasmidpCB1926 using a partial digest (as there is a second EcoRI site withinthe Pi-ta cDNA nucleic acid fragment) and cloned into the same sites ofplasmid Litmus 28a (New England Biolabs). Plasmid pCB2021 was thendigested with AgeI and KpnI and a 501 bp AgeI-KpnI In2-1 terminatornucleic acid fragment was cloned into the same sites to create pCB2022.The In2-1 terminator sequence may be amplified from plasmids containingIn2-1 terminator sequence (e.g., pJE514, pJE516, and pTDS136 disclosedin U.S. Pat. No. 5,364,780, the disclosure of which is herebyincorporated by reference) using primers GB188 (SEQ ID NO:54) and GB189(SEQ ID NO:55), then digested with AgeI and KpnI.

GB188: 5′-GCCGACCGGTAGATCTGACAAAGCAGCATTAG-3′ (SEQ ID NO:54)

GB189: 5′-CGGCGGTACCGCTCTCTCTCTCCCCTTGC-3′ (SEQ ID NO:55)

Modified Pi-ta nucleic acid fragments identified in Example 5 whichencode Pi-ta protein wherein the amino acid alteration at position 918is selected from the group consisting of M, C, I, R, K, N, L and Q, andare suitable for stable rice transformation can be made in a two-stagecloning step. A 2.1 kb BamHI-HindIII fragment from pCB2176, pCB2180,pCB2177, pCB2173, pCB2174, pCB2175, pCB2185, or pCB2183 can be clonedinto vector pCB2021 to replace the corresponding fragment from thewild-type Pi-ta nucleic acid fragment present in pCB2021. Then the 501bp AgeI-KpnI In2-1 terminator nucleic acid fragment can be excised fromvector pCB2022 by digesting with AgeI and KpnI and cloned into the samesites on the new vector that has also been digested with AgeI and KpnI.These new vectors can then be used in stable rice transformation asdescribed in Example 7 below.

Example 7 Testing Chimeric Genes Encoding Modified Pi-ta Proteins fromExample 6 by Stable Rice Transformation

The bacterial hygromycin B phosphotransferase (Hpt II) gene fromStreptomyces hygroscopicus that confers resistance to the antibiotic canbe used as the selectable marker for rice transformation. In the vectorthat could be used, pML 18, the Hpt II gene has been engineered with the35S promoter from Cauliflower Mosaic Virus and the termination andpolyadenylation signals from the octopine synthase gene of Agrobacteriumtumefaciens. pML 18 is described in WO 97/47731, which was published onDec. 18, 1997, the disclosure of which is hereby incorporated byreference.

Embryogenic callus cultures derived from the scutellum of germinatingNipponbare seeds can serve as source material for transformationexperiments. This material is generated by germinating sterile riceseeds on a callus initiation media (MS salts, Nitsch and Nitschvitamins, 1.0 mg/l 2,4-D and 10 μM AgNO₃) in the dark at 27-28° C.Embryogenic callus proliferating from the scutellum of the embryos arethen transferred to CM media (N6 salts, Nitsch and Nitsch vitamins, 1mg/l 2,4-D, Chu et al., 1985, Sci. Sinica 18: 659-668). Callus culturescan be maintained on CM by routine sub-culture at two week intervals andused for transformation within 10 weeks of initiation.

Callus can be prepared for transformation by subculturing 0.5-1.0 mmpieces approximately 1 mm apart, arranged in a circular area of about 4cm in diameter, in the center of a circle of Whatman #541 paper placedon CM media. The plates with callus are then incubated in the dark at27-28° C. for 3-5 days. Prior to bombardment, the filters with callusare transferred to CM supplemented with 0.25 M mannitol and 0.25 Msorbitol for 3 hr. in the dark. The petri dish lids are then left ajarfor 20-45 minutes in a sterile hood to allow moisture on tissue todissipate.

Circular plasmid DNA from two different plasmids, for example, pML18containing the selectable marker for rice transformation and a vectorcontaining the modified Pi-ta nucleic acid fragment, can beco-precipitated onto the surface of gold particles. To accomplish this,a total of 10 μg of DNA at a 2:1 ratio of trait:selectable marker DNAsare added to 50 μl aliquot of gold particles that have been resuspendedat a concentration of 60 mg ml⁻¹. Calcium chloride (50 μl of a 2.5 Msolution) and spermidine (20 μl of a 0.1 M solution) are then added tothe gold-DNA suspension and the tube would be vortexed for 3 min. Thegold particles are centrifuged in a microfuge for 1 sec and thesupernatant removed. The gold particles are then washed twice with 1 mlof absolute ethanol and then resuspended in 50 μl of absolute ethanoland sonicated (bath sonicator) for one second to disperse the goldparticles. The gold suspension is then incubated at −70° C. for fiveminutes and sonicated (bath sonicator) if needed to disperse theparticles. Six μl of the DNA-coated gold particles can then be loadedonto mylar macrocarrier disks and the ethanol allowed to evaporate.

At the end of the drying period, a petri dish containing the tissue isplaced in the chamber of the PDS-1000/He. The air in the chamber is thenevacuated to a vacuum of 28-29 inches Hg. The macrocarrier acceleratedwith a helium shock wave using a rupture membrane that bursts when theHe pressure in the shock tube reaches 1080-1100 psi. The tissue isplaced approximately 8 cm from the stopping screen and the callusbombarded two times. Five to seven plates of tissue are bombarded inthis way with the DNA-coated gold particles. Following bombardment, thecallus tissue is transferred to CM media without supplemental sorbitolor mannitol.

Within 3-5 days after bombardment the callus tissue is transferred to SMmedia (CM medium containing 50 mg/I hygromycin). To accomplish this,callus tissue is transferred from plates to sterile 50 ml conical tubesand weighed. Molten top-agar at 40° C. can be added using 2.5 ml of topagar/100 mg of callus. Callus clumps are broken into fragments of lessthan 2 mm diameter by repeated dispensing through a 10 ml pipet. Threeml aliquots of the callus suspension are plated onto fresh SM media andthe plates can be incubated in the dark for 4 weeks at 27-28° C. After 4weeks, transgenic callus events are identified, transferred to fresh SMplates and grown for an additional 2 weeks in the dark at 27-28° C.

Growing callus is transferred to RM1 media (MS salts, Nitsch and Nitschvitamins, 2% sucrose, 3% sorbitol, 0.4% gelrite+50 ppm hyg B) for 2weeks in the dark at 25° C. After 2 weeks the callus can be transferredto RM2 media (MS salts, Nitsch and Nitsch vitamins, 3% sucrose, 0.4%gelrite+50 ppm hyg B) and placed under cool white light (˜40 μEm⁻²s⁻¹)with a 12 hr photoperiod at 25° C. and 30-40% humidity. After 2-4 weeksin the light, callus begins to organize, and form shoots. Shoots arethen removed from surrounding callus/media and gently transferred to RM3media (½×MS salts, Nitsch and Nitsch vitamins, 1% sucrose+50 ppmhygromycin B) in phytatrays (Sigma Chemical Co., St. Louis, Mo.) andincubation is continued using the same conditions as described in theprevious step.

Plants are transferred from RM3 to 4″ pots containing Metro mix 350after 2-3 weeks, when sufficient root and shoot growth had occurred.Plants can be grown using a 12 hr/12 hr light/dark cycle using ˜30/18°C. day/night temperature regimen.

                   #             SEQUENCE LISTING<160> NUMBER OF SEQ ID NOS: 60 <210> SEQ ID NO 1 <211> LENGTH: 4119<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial  #Sequence:Plasmid<400> SEQUENCE: 1cacctaaatt gtaagcgtta atattttgtt aaaattcgcg ttaaattttt gt#taaatcag     60ctcatttttt aaccaatagg ccgaaatcgg caaaatccct tataaatcaa aa#gaatagac    120cgagataggg ttgagtgttg ttccagtttg gaacaagagt ccactattaa ag#aacgtgga    180ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat ggcccactac gt#gaaccatc    240accctaatca agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga ac#cctaaagg    300gagcccccga tttagagctt gacggggaaa gccggcgaac gtggcgagaa ag#gaagggaa    360gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc tg#cgcgtaac    420caccacaccc gccgcgctta atgcgccgct acagggcgcg tcccattcgc ca#ttcaggct    480gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc ag#ctggcgaa    540agggggatgt gctgcaaggc gattaagttg ggtaacgcca gggttttccc ag#tcacgacg    600ttgtaaaacg acggccagtg aattgtaata cgactcacta tagggcgaat tg#ggtaccgg    660gccccccctc gaggtcgatc cgagggtagg tctaggggcc tgatcctcac aa#tatttttg    720taaatttcaa aagtcaggga gcatgaatta tgtagttatt aataatatgg gc#ccaactct    780taccttatat aaaattgtgg atgatatact aataaaagtg gacctaatta cc#tgcataat    840aatgcagata attaacacta gcaaaatata attcgataat attattaatg ct#aaataacg    900cattaataaa ccaaataagt tttacatctt cctaaagctt tgaaaaaagt ca#agctgaaa    960taataaataa gttggcgttg ttataaaatc gacccgtttc cgcctttatt gg#tttaattc   1020ggatagagaa cattttgctt ataattccaa acatacaaac aattatccac tg#actgaaaa   1080tcgacagttt tgtttgcaca atcaacatta taattacaat taaaaacttc tg#cacaatta   1140acattatttt tgcatcgatg cttttttatt cattattttt ttttcacacc gt#tgcgattt   1200cggccttcac caacattggc accttttcac acccagttta cgattacaat cc#aattccaa   1260accatatcca cggagattta aaaaggcggg cttatattga acgctattcc ca#atgttcag   1320attcgcaggc ctccgaaatt cgtgccgcgc taaaaagttg tgccgagctc gc#ctcgtggg   1380gctatcacgc cgttaaaaat gacaatcggt tatttagatt aatctttaaa ac#tgacagca   1440cagatattca aaactgggtt caaaagaatt ttaacgaaat ttacaaggaa tg#taacaggg   1500acgcggacga aatttctcta acctgccacg ataaaaatgt ttatacgtgc gt#ccgagaag   1560gagttcataa tttggcgtat gcacttatta acgaaaaaga aattgttata tg#ccctcctt   1620tcttcaacaa ccccgtaaac agcagggaaa ttactgccgg taaccaagat ac#agttatat   1680tacatgaaat ggtgcatata attttaaaag agtggaaaga ttatggttac ga#atgggatg   1740ggattcacaa attggatagt acagaaagta ttaaaaaccc cgacagttat gc#tatttttg   1800cacaatgtgc acgttataaa tattgttaaa taacgttagt gttggaatgg ag#ggaatcgc   1860ggtcaatcta acaatagagg gatccactag ttctagagcg gccgccaccg cg#gtggagct   1920ccagcttttg ttccctttag tgagggttaa tttcgagctt ggcgtaatca tg#gtcatagc   1980tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga gc#cggaagca   2040taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt gc#gttgcgct   2100cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga at#cggccaac   2160gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc ac#tgactcgc   2220tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gt#aatacggt   2280tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc ca#gcaaaagg   2340ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc cc#ccctgacg   2400agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ct#ataaagat   2460accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ct#gccgctta   2520ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat ag#ctcacgct   2580gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg ca#cgaacccc   2640ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aa#cccggtaa   2700gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gc#gaggtatg   2760taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact ag#aaggacag   2820tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt gg#tagctctt   2880gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag ca#gcagatta   2940cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tc#tgacgctc   3000agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa ag#gatcttca   3060cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata ta#tgagtaaa   3120cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg at#ctgtctat   3180ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata cg#ggagggct   3240taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gc#tccagatt   3300tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gc#aactttat   3360ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt tc#gccagtta   3420atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tc#gtcgtttg   3480gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga tc#ccccatgt   3540tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aa#gttggccg   3600cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc at#gccatccg   3660taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa ta#gtgtatgc   3720ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca ca#tagcagaa   3780ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca ag#gatcttac   3840cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct tc#agcatctt   3900ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gc#aaaaaagg   3960gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa ta#ttattgaa   4020gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt ta#gaaaaata   4080 aacaaatagg ggttccgcgc acatttcccc gaaaagtgc      #                   #  4119 <210> SEQ ID NO 2 <211> LENGTH: 672<212> TYPE: DNA <213> ORGANISM: Magnaporthe grisea <400> SEQUENCE: 2atgctttttt attcattatt tttttttcac accgttgcga tttcggcctt ca#ccaacatt     60ggcacctttt cacacccagt ttacgattac aatccaattc caaaccatat cc#acggagat    120ttaaaaaggc gggcttatat tgaacgctat tcccaatgtt cagattcgca gg#cctccgaa    180attcgtgccg cgctaaaaag ttgtgccgag ctcgcctcgt ggggctatca cg#ccgttaaa    240aatgacaatc ggttatttag attaatcttt aaaactgaca gcacagatat tc#aaaactgg    300gttcaaaaga attttaacga aatttacaag gaatgtaaca gggacgcgga cg#aaatttct    360ctaacctgcc acgataaaaa tgtttatacg tgcgtccgag aaggagttca ta#atttggcg    420tatgcactta ttaacgaaaa agaaattgtt atatgccctc ctttcttcaa ca#accccgta    480aacagcaggg aaattactgc cggtaaccaa gatacagtta tattacatga aa#tggtgcat    540ataattttaa aagagtggaa agattatggt tacgaatggg atgggattca ca#aattggat    600agtacagaaa gtattaaaaa ccccgacagt tatgctattt ttgcacaatg tg#cacgttat    660 aaatattgtt aa               #                  #                   #      672 <210> SEQ ID NO 3 <211> LENGTH: 223<212> TYPE: PRT <213> ORGANISM: Magnaporthe grisea <400> SEQUENCE: 3Met Leu Phe Tyr Ser Leu Phe Phe Phe His Th #r Val Ala Ile Ser Ala  1               5  #                 10  #                 15Phe Thr Asn Ile Gly Thr Phe Ser His Pro Va #l Tyr Asp Tyr Asn Pro             20      #             25      #             30Ile Pro Asn His Ile His Gly Asp Leu Lys Ar #g Arg Ala Tyr Ile Glu         35          #         40          #         45Arg Tyr Ser Gln Cys Ser Asp Ser Gln Ala Se #r Glu Ile Arg Ala Ala     50              #     55              #     60Leu Lys Ser Cys Ala Glu Leu Ala Ser Trp Gl #y Tyr His Ala Val Lys 65                  # 70                  # 75                  # 80Asn Asp Asn Arg Leu Phe Arg Leu Ile Phe Ly #s Thr Asp Ser Thr Asp                 85  #                 90  #                 95Ile Gln Asn Trp Val Gln Lys Asn Phe Asn Gl #u Ile Tyr Lys Glu Cys            100       #           105       #           110Asn Arg Asp Ala Asp Glu Ile Ser Leu Thr Cy #s His Asp Lys Asn Val        115           #       120           #       125Tyr Thr Cys Val Arg Glu Gly Val His Asn Le #u Ala Tyr Ala Leu Ile    130               #   135               #   140Asn Glu Lys Glu Ile Val Ile Cys Pro Pro Ph #e Phe Asn Asn Pro Val145                 1 #50                 1 #55                 1 #60Asn Ser Arg Glu Ile Thr Ala Gly Asn Gln As #p Thr Val Ile Leu His                165   #               170   #               175Glu Met Val His Ile Ile Leu Lys Glu Trp Ly #s Asp Tyr Gly Tyr Glu            180       #           185       #           190Trp Asp Gly Ile His Lys Leu Asp Ser Thr Gl #u Ser Ile Lys Asn Pro        195           #       200           #       205Asp Ser Tyr Ala Ile Phe Ala Gln Cys Ala Ar #g Tyr Lys Tyr Cys    210               #   215               #   220 <210> SEQ ID NO 4<211> LENGTH: 788 <212> TYPE: DNA <213> ORGANISM: Magnaporthe grisea<400> SEQUENCE: 4ttatttttat cgatatgctt tttgattcat tatttttttt tcacaccgtt gc#gatttcgg     60cctgcaccaa cattggcacc ttttcacacc cagtttacga ttacaatcca at#tccaaacc    120atatccacgg agatttaaaa aggcgggctt atattgaacg ctattcccaa tg#ttcagatt    180cgcaggcctc cgaaattcgt gccgcgctaa aaagttgcgc cgagctcgcc tc#gtggggct    240atcacgccgt taaaagtaac aatcggttat ttaaattaat ctttaaaact ga#cagcacag    300atattcaaaa ctgggttcaa aataatttta acgaaattta caaggaatgt aa#cagggacg    360cggacgaaat ttctctaacc tgccacgata aaaatgttta tacgtgcgtc cg#acaaggag    420ttcataattt ggcgtatgca cttattaacg aaaaagaaat tgttatatgc cc#tcctttct    480tcaacaaccc cgtaaacagc agggaaatta ctgccggtaa ccaagataca at#tatattac    540atgaaatggt gcatataatt ttaaaagagt ggaaagatta tggttgcgaa tg#ggatggga    600ttcacaaatt ggatagtaca gaaagtatta aaaaccccga cagttatgct at#ttttgcac    660aatgtgcacg ttataaatat tgttaaataa cgttagtgtt ggaatggagg ga#atcgcggt    720caatctaaca atagaggggg atccactagt tctagagcgg ccgccaccgc gg#tggagctc    780 cagctttt                 #                  #                   #         788 <210> SEQ ID NO 5 <211> LENGTH: 223<212> TYPE: PRT <213> ORGANISM: Magnaporthe grisea <400> SEQUENCE: 5Met Leu Phe Asp Ser Leu Phe Phe Phe His Th #r Val Ala Ile Ser Ala  1               5  #                 10  #                 15Cys Thr Asn Ile Gly Thr Phe Ser His Pro Va #l Tyr Asp Tyr Asn Pro             20      #             25      #             30Ile Pro Asn His Ile His Gly Asp Leu Lys Ar #g Arg Ala Tyr Ile Glu         35          #         40          #         45Arg Tyr Ser Gln Cys Ser Asp Ser Gln Ala Se #r Glu Ile Arg Ala Ala     50              #     55              #     60Leu Lys Ser Cys Ala Glu Leu Ala Ser Trp Gl #y Tyr His Ala Val Lys 65                  # 70                  # 75                  # 80Ser Asn Asn Arg Leu Phe Lys Leu Ile Phe Ly #s Thr Asp Ser Thr Asp                 85  #                 90  #                 95Ile Gln Asn Trp Val Gln Asn Asn Phe Asn Gl #u Ile Tyr Lys Glu Cys            100       #           105       #           110Asn Arg Asp Ala Asp Glu Ile Ser Leu Thr Cy #s His Asp Lys Asn Val        115           #       120           #       125Tyr Thr Cys Val Arg Gln Gly Val His Asn Le #u Ala Tyr Ala Leu Ile    130               #   135               #   140Asn Glu Lys Glu Ile Val Ile Cys Pro Pro Ph #e Phe Asn Asn Pro Val145                 1 #50                 1 #55                 1 #60Asn Ser Arg Glu Ile Thr Ala Gly Asn Gln As #p Thr Ile Ile Leu His                165   #               170   #               175Glu Met Val His Ile Ile Leu Lys Glu Trp Ly #s Asp Tyr Gly Cys Glu            180       #           185       #           190Trp Asp Gly Ile His Lys Leu Asp Ser Thr Gl #u Ser Ile Lys Asn Pro        195           #       200           #       205Asp Ser Tyr Ala Ile Phe Ala Gln Cys Ala Ar #g Tyr Lys Tyr Cys    210               #   215               #   220 <210> SEQ ID NO 6<211> LENGTH: 672 <212> TYPE: DNA <213> ORGANISM: Magnaporthe grisea<400> SEQUENCE: 6atgctttttt attcattatt tttttttcac accgttgcga tttcggcctt ca#ccaacatt     60ggcacctttt cacacccagt ttacgattac aatccaattc caaaccatat cc#acggagat    120ttaaaaaggc gggcttatat tgaacgctat tcccaatgtt cagattcgca gg#cctccgaa    180attcgtgccg cgctaaaaag ttgcgccgag ctcgcctcgt ggggctatca cg#ccgttaaa    240agtaacaatc ggttatttaa attaatcttt aaaactgaca gcacagatat tc#aaaactgg    300gttcaaaata attttaacga aatttacaag gaatgtaaca gggacgcgga cg#aaatttct    360ctaacctgcc acgataaaaa tgtttatacg tgcgtccgag aaggagttca ta#atttggcg    420tatgcactta ttaacgaaaa agaaattgtt atatgccctc ctttcttcaa ca#accccgta    480aacagcaggg aaattactgc cggtaaccaa gatacaatta tattacatga aa#tggtgcat    540ataattttaa aagagtggaa agattatggt tgcgaatggg atgggattca ca#aattggat    600agtacagaaa gtattaaaaa ccccgacagt tatgctattt ttgcacaatg tg#cacgttat    660 aaatattgtt aa               #                  #                   #      672 <210> SEQ ID NO 7 <211> LENGTH: 223<212> TYPE: PRT <213> ORGANISM: Magnaporthe grisea <400> SEQUENCE: 7Met Leu Phe Tyr Ser Leu Phe Phe Phe His Th #r Val Ala Ile Ser Ala  1               5  #                 10  #                 15Phe Thr Asn Ile Gly Thr Phe Ser His Pro Va #l Tyr Asp Tyr Asn Pro             20      #             25      #             30Ile Pro Asn His Ile His Gly Asp Leu Lys Ar #g Arg Ala Tyr Ile Glu         35          #         40          #         45Arg Tyr Ser Gln Cys Ser Asp Ser Gln Ala Se #r Glu Ile Arg Ala Ala     50              #     55              #     60Leu Lys Ser Cys Ala Glu Leu Ala Ser Trp Gl #y Tyr His Ala Val Lys 65                  # 70                  # 75                  # 80Ser Asn Asn Arg Leu Phe Lys Leu Ile Phe Ly #s Thr Asp Ser Thr Asp                 85  #                 90  #                 95Ile Gln Asn Trp Val Gln Asn Asn Phe Asn Gl #u Ile Tyr Lys Glu Cys            100       #           105       #           110Asn Arg Asp Ala Asp Glu Ile Ser Leu Thr Cy #s His Asp Lys Asn Val        115           #       120           #       125Tyr Thr Cys Val Arg Glu Gly Val His Asn Le #u Ala Tyr Ala Leu Ile    130               #   135               #   140Asn Glu Lys Glu Ile Val Ile Cys Pro Pro Ph #e Phe Asn Asn Pro Val145                 1 #50                 1 #55                 1 #60Asn Ser Arg Glu Ile Thr Ala Gly Asn Gln As #p Thr Ile Ile Leu His                165   #               170   #               175Glu Met Val His Ile Ile Leu Lys Glu Trp Ly #s Asp Tyr Gly Cys Glu            180       #           185       #           190Trp Asp Gly Ile His Lys Leu Asp Ser Thr Gl #u Ser Ile Lys Asn Pro        195           #       200           #       205Asp Ser Tyr Ala Ile Phe Ala Gln Cys Ala Ar #g Tyr Lys Tyr Cys    210               #   215               #   220 <210> SEQ ID NO 8<211> LENGTH: 884 <212> TYPE: DNA <213> ORGANISM: Magnaporthe grisea<400> SEQUENCE: 8atgctttttt attcattgtt atttttattt cacaccgttg cgatttcggc ct#tcaccaac     60attggcacct tttcacaccc agtttacgat tacaatccaa ttccaaacca ta#tccacgga    120gatttaaaaa ggcgggctta tattgaacgc tattcccaat gttcagattc gc#aggcctcc    180gaaattcgtg ccgcgctaaa aaggtaaatt gaaactcttt aaaacaaatt cg#gaaaacaa    240tgttaaatat tttgtttagt tgcgccgagc tcgcctcgtg gggctatcac gc#cgttaaaa    300gtgacaatcg gttatttaaa ttaatcttta aaactgacag cacagatatt ca#aaactggg    360ttcaaaataa ttttaacgaa atttacaagg aatgtaacag ggacgcggac ga#aatttctc    420taacctgcca cgataaaaat gtttatacgt gcgtccgaga agaagttcat aa#tttggcgt    480atgcacttat taacgaaaaa gaaattgtta tatgccctcc tttcttcaac aa#ccccgtaa    540acagcaggga aattactgcc ggtaaccaag atacaattat attacatgaa at#ggtgcata    600taattttaag taagtttgct tttacaaatt gataaaacat ttacaaaagt tt#atttataa    660aaattttcaa aaactaaaaa ttcaaatttt tatttagaag agtggaaaga tt#atggttac    720gaatgggatg ggattcacaa gtaagttgtc gaaaaacaaa atgctgaatg tt#gttttata    780ttgataaatt ctaattaata ttaagattgg atagtacaga aagtattaaa aa#ccccgaca    840 gttatgctat ttttgcacaa tgtgcacgtt ataaatattg ttaa   #                   #884 <210> SEQ ID NO 9 <211> LENGTH: 675<212> TYPE: DNA <213> ORGANISM: Magnaporthe grisea <400> SEQUENCE: 9atgctttttt attcattgtt atttttattt cacaccgttg cgatttcggc ct#tcaccaac     60attggcacct tttcacaccc agtttacgat tacaatccaa ttccaaacca ta#tccacgga    120gatttaaaaa ggcgggctta tattgaacgc tattcccaat gttcagattc gc#aggcctcc    180gaaattcgtg ccgcgctaaa aagttgcgcc gagctcgcct cgtggggcta tc#acgccgtt    240aaaagtgaca atcggttatt taaattaatc tttaaaactg acagcacaga ta#ttcaaaac    300tgggttcaaa ataattttaa cgaaatttac aaggaatgta acagggacgc gg#acgaaatt    360tctctaacct gccacgataa aaatgtttat acgtgcgtcc gagaagaagt tc#ataatttg    420gcgtatgcac ttattaacga aaaagaaatt gttatatgcc ctcctttctt ca#acaacccc    480gtaaacagca gggaaattac tgccggtaac caagatacaa ttatattaca tg#aaatggtg    540catataattt taaaagagtg gaaagattat ggttacgaat gggatgggat tc#acaaattg    600gatagtacag aaagtattaa aaaccccgac agttatgcta tttttgcaca at#gtgcacgt    660 tataaatatt gttaa               #                  #                   #   675 <210> SEQ ID NO 10 <211> LENGTH: 224<212> TYPE: PRT <213> ORGANISM: Magnaporthe grisea <400> SEQUENCE: 10Met Leu Phe Tyr Ser Leu Leu Phe Leu Phe Hi #s Thr Val Ala Ile Ser  1               5  #                 10  #                 15Ala Phe Thr Asn Ile Gly Thr Phe Ser His Pr #o Val Tyr Asp Tyr Asn             20      #             25      #             30Pro Ile Pro Asn His Ile His Gly Asp Leu Ly #s Arg Arg Ala Tyr Ile         35          #         40          #         45Glu Arg Tyr Ser Gln Cys Ser Asp Ser Gln Al #a Ser Glu Ile Arg Ala     50              #     55              #     60Ala Leu Lys Ser Cys Ala Glu Leu Ala Ser Tr #p Gly Tyr His Ala Val 65                  # 70                  # 75                  # 80Lys Ser Asp Asn Arg Leu Phe Lys Leu Ile Ph #e Lys Thr Asp Ser Thr                 85  #                 90  #                 95Asp Ile Gln Asn Trp Val Gln Asn Asn Phe As #n Glu Ile Tyr Lys Glu            100       #           105       #           110Cys Asn Arg Asp Ala Asp Glu Ile Ser Leu Th #r Cys His Asp Lys Asn        115           #       120           #       125Val Tyr Thr Cys Val Arg Glu Glu Val His As #n Leu Ala Tyr Ala Leu    130               #   135               #   140Ile Asn Glu Lys Glu Ile Val Ile Cys Pro Pr #o Phe Phe Asn Asn Pro145                 1 #50                 1 #55                 1 #60Val Asn Ser Arg Glu Ile Thr Ala Gly Asn Gl #n Asp Thr Ile Ile Leu                165   #               170   #               175His Glu Met Val His Ile Ile Leu Lys Glu Tr #p Lys Asp Tyr Gly Tyr            180       #           185       #           190Glu Trp Asp Gly Ile His Lys Leu Asp Ser Th #r Glu Ser Ile Lys Asn        195           #       200           #       205Pro Asp Ser Tyr Ala Ile Phe Ala Gln Cys Al #a Arg Tyr Lys Tyr Cys    210               #   215               #   220 <210> SEQ ID NO 11<211> LENGTH: 884 <212> TYPE: DNA <213> ORGANISM: Magnaporthe grisea<400> SEQUENCE: 11atgctttttt attcattgtt atttttattt cacaccgttg cgatttcggc ct#tcaccaac     60attggcacct tttcacaccc agtttacgat tacaatccaa ttccaaacca ta#tccacgga    120gatttaaaaa ggcgggctta tattgaacgc tattcccaat gttcagattc gc#aggcctcc    180gaaattcgtg ccgcgctaaa aaggtaaatt gaaactcttt aaaacaaatt cg#gaaaacaa    240tgttaaatat tttgtttagt tgcgccgagc tcgcctcgtg gggctatcac gc#cgttaaaa    300gtaacaatcg gttatttaaa ttaatcttta aaactgacag cacagatatt ca#aaactggg    360ttcaaaataa ttttaacgaa atttacaagg aatgtaacag ggacgcggac ga#aatttctc    420taacctgcca cgataaaaat gtttatacgt gcgtccgaga agaagttcat aa#tttggcgt    480atgcacttat taacgaaaaa gaaattgtta tatgccctcc tttcttcaac aa#ccccgtaa    540acagcaggga aattactgcc ggtaaccaag atacaattat attacatgaa at#ggtgcata    600taattttaag taagtttgct tttacaaatt gataaaacat ttacaaaagt tt#atttataa    660aaattttcaa aaactaaaaa ttcaaatttt tatttagaag agtggaaaga tt#atggttgc    720gaatgggatg ggattcacaa gtaagttgtc gaaaaacaaa atgctgaatg tt#gttttata    780ttgataaatt ctaattaata ttaagattgg atagtacaga aagtattaaa aa#ccccgaca    840 gttatgctat ttttgcacaa tgtgcacgtt ataaatattg ttaa   #                   #884 <210> SEQ ID NO 12 <211> LENGTH: 675<212> TYPE: DNA <213> ORGANISM: Magnaporthe grisea <400> SEQUENCE: 12atgctttttt attcattgtt atttttattt cacaccgttg cgatttcggc ct#tcaccaac     60attggcacct tttcacaccc agtttacgat tacaatccaa ttccaaacca ta#tccacgga    120gatttaaaaa ggcgggctta tattgaacgc tattcccaat gttcagattc gc#aggcctcc    180gaaattcgtg ccgcgctaaa aagttgcgcc gagctcgcct cgtggggcta tc#acgccgtt    240aaaagtaaca atcggttatt taaattaatc tttaaaactg acagcacaga ta#ttcaaaac    300tgggttcaaa ataattttaa cgaaatttac aaggaatgta acagggacgc gg#acgaaatt    360tctctaacct gccacgataa aaatgtttat acgtgcgtcc gagaagaagt tc#ataatttg    420gcgtatgcac ttattaacga aaaagaaatt gttatatgcc ctcctttctt ca#acaacccc    480gtaaacagca gggaaattac tgccggtaac caagatacaa ttatattaca tg#aaatggtg    540catataattt taaaagagtg gaaagattat ggttgcgaat gggatgggat tc#acaaattg    600gatagtacag aaagtattaa aaaccccgac agttatgcta tttttgcaca at#gtgcacgt    660 tataaatatt gttaa               #                  #                   #   675 <210> SEQ ID NO 13 <211> LENGTH: 224<212> TYPE: PRT <213> ORGANISM: Magnaporthe grisea <400> SEQUENCE: 13Met Leu Phe Tyr Ser Leu Leu Phe Leu Phe Hi #s Thr Val Ala Ile Ser  1               5  #                 10  #                 15Ala Phe Thr Asn Ile Gly Thr Phe Ser His Pr #o Val Tyr Asp Tyr Asn             20      #             25      #             30Pro Ile Pro Asn His Ile His Gly Asp Leu Ly #s Arg Arg Ala Tyr Ile         35          #         40          #         45Glu Arg Tyr Ser Gln Cys Ser Asp Ser Gln Al #a Ser Glu Ile Arg Ala     50              #     55              #     60Ala Leu Lys Ser Cys Ala Glu Leu Ala Ser Tr #p Gly Tyr His Ala Val 65                  # 70                  # 75                  # 80Lys Ser Asn Asn Arg Leu Phe Lys Leu Ile Ph #e Lys Thr Asp Ser Thr                 85  #                 90  #                 95Asp Ile Gln Asn Trp Val Gln Asn Asn Phe As #n Glu Ile Tyr Lys Glu            100       #           105       #           110Cys Asn Arg Asp Ala Asp Glu Ile Ser Leu Th #r Cys His Asp Lys Asn        115           #       120           #       125Val Tyr Thr Cys Val Arg Glu Glu Val His As #n Leu Ala Tyr Ala Leu    130               #   135               #   140Ile Asn Glu Lys Glu Ile Val Ile Cys Pro Pr #o Phe Phe Asn Asn Pro145                 1 #50                 1 #55                 1 #60Val Asn Ser Arg Glu Ile Thr Ala Gly Asn Gl #n Asp Thr Ile Ile Leu                165   #               170   #               175His Glu Met Val His Ile Ile Leu Lys Glu Tr #p Lys Asp Tyr Gly Cys            180       #           185       #           190Glu Trp Asp Gly Ile His Lys Leu Asp Ser Th #r Glu Ser Ile Lys Asn        195           #       200           #       205Pro Asp Ser Tyr Ala Ile Phe Ala Gln Cys Al #a Arg Tyr Lys Tyr Cys    210               #   215               #   220 <210> SEQ ID NO 14<211> LENGTH: 884 <212> TYPE: DNA <213> ORGANISM: Magnaporthe grisea<400> SEQUENCE: 14atgctttttt attcatttat attttatttt cacaccgttg caatttcggc ct#tcaccaac     60attggcacct tttcataccc agtttacatt tacaatccaa ttccaaacca ta#tccacgga    120gatttaaaaa ggcgggctta tattgaaccc tattcccaat gttcaaattc gc#aggactcc    180gaaattcgtg ccgcgctaaa aaggtaaatt gaaattcttt aagacaaatt cg#gaaaacaa    240tgttaaatat tttgtttagt tgtgccgaac tcgcctcgtg ggcctatcac gc#cgttgaaa    300atgacaatcg gttatttgaa ttgattttta aaactgacag cacaaatatt aa#aaactggg    360ttcaaaataa ttttaacgaa attcacaagg aatgtaacag ggacgcggac ga#aatttctc    420tatcctgcca cgatacaagt gtttatacgt gcgtccgaga aggagttcat ct#tttgggct    480atgcaaagat gtacgaaaaa caagttgttt tatgccctca tttctttgat ca#ccccgtaa    540acagcaggga aatcactgcc caaaaccaag atacagttat attgcatgaa at#gctgcata    600taattctaag taagtttgct tttacaaatt aataaaatct ttacaaaagg tt#attcataa    660atattttcaa aaactaacaa ttcaaatttt tatttagatg agtgggaaga tt#atggttac    720gaatgggatg ggattcacaa gtaagttgtc gaaaaacaaa tttgctaaaa tt#attttata    780ttgataaatt ctaattaata taaagtttgg atagtacaac aagtattaaa aa#ccccgaca    840 gctatgctat ttttgcacaa tgtgcacgtt ataaatattg ttaa   #                   #884 <210> SEQ ID NO 15 <211> LENGTH: 675<212> TYPE: DNA <213> ORGANISM: Magnaporthe grisea <400> SEQUENCE: 15atgctttttt attcatttat attttatttt cacaccgttg caatttcggc ct#tcaccaac     60attggcacct tttcataccc agtttacatt tacaatccaa ttccaaacca ta#tccacgga    120gatttaaaaa ggcgggctta tattgaaccc tattcccaat gttcaaattc gc#aggactcc    180gaaattcgtg ccgcgctaaa aagttgtgcc gaactcgcct cgtgggccta tc#acgccgtt    240gaaaatgaca atcggttatt tgaattgatt tttaaaactg acagcacaaa ta#ttaaaaac    300tgggttcaaa ataattttaa cgaaattcac aaggaatgta acagggacgc gg#acgaaatt    360tctctatcct gccacgatac aagtgtttat acgtgcgtcc gagaaggagt tc#atcttttg    420ggctatgcaa agatgtacga aaaacaagtt gttttatgcc ctcatttctt tg#atcacccc    480gtaaacagca gggaaatcac tgcccaaaac caagatacag ttatattgca tg#aaatgctg    540catataattc taaatgagtg ggaagattat ggttacgaat gggatgggat tc#acaatttg    600gatagtacaa caagtattaa aaaccccgac agctatgcta tttttgcaca at#gtgcacgt    660 tataaatatt gttaa               #                  #                   #   675 <210> SEQ ID NO 16 <211> LENGTH: 224<212> TYPE: PRT <213> ORGANISM: Magnaporthe grisea <400> SEQUENCE: 16Met Leu Phe Tyr Ser Phe Ile Phe Tyr Phe Hi #s Thr Val Ala Ile Ser  1               5  #                 10  #                 15Ala Phe Thr Asn Ile Gly Thr Phe Ser Tyr Pr #o Val Tyr Ile Tyr Asn             20      #             25      #             30Pro Ile Pro Asn His Ile His Gly Asp Leu Ly #s Arg Arg Ala Tyr Ile         35          #         40          #         45Glu Pro Tyr Ser Gln Cys Ser Asn Ser Gln As #p Ser Glu Ile Arg Ala     50              #     55              #     60Ala Leu Lys Ser Cys Ala Glu Leu Ala Ser Tr #p Ala Tyr His Ala Val 65                  # 70                  # 75                  # 80Glu Asn Asp Asn Arg Leu Phe Glu Leu Ile Ph #e Lys Thr Asp Ser Thr                 85  #                 90  #                 95Asn Ile Lys Asn Trp Val Gln Asn Asn Phe As #n Glu Ile His Lys Glu            100       #           105       #           110Cys Asn Arg Asp Ala Asp Glu Ile Ser Leu Se #r Cys His Asp Thr Ser        115           #       120           #       125Val Tyr Thr Cys Val Arg Glu Gly Val His Le #u Leu Gly Tyr Ala Lys    130               #   135               #   140Met Tyr Glu Lys Gln Val Val Leu Cys Pro Hi #s Phe Phe Asp His Pro145                 1 #50                 1 #55                 1 #60Val Asn Ser Arg Glu Ile Thr Ala Gln Asn Gl #n Asp Thr Val Ile Leu                165   #               170   #               175His Glu Met Leu His Ile Ile Leu Asn Glu Tr #p Glu Asp Tyr Gly Tyr            180       #           185       #           190Glu Trp Asp Gly Ile His Asn Leu Asp Ser Th #r Thr Ser Ile Lys Asn        195           #       200           #       205Pro Asp Ser Tyr Ala Ile Phe Ala Gln Cys Al #a Arg Tyr Lys Tyr Cys    210               #   215               #   220 <210> SEQ ID NO 17<211> LENGTH: 40 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Description of Artificial #Sequence:Synthetic       Oligonucleotide <400> SEQUENCE: 17gatcgaatcg atatgctttt ttattcatta tttttttttc      #                  #    40 <210> SEQ ID NO 18 <211> LENGTH: 33 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial  #Sequence:Synthetic      Oligonucleotide <400> SEQUENCE: 18gatcgaggat ccccctctat tgttagattg acc        #                  #         33 <210> SEQ ID NO 19 <211> LENGTH: 34 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial  #Sequence:Synthetic      Oligonucleotide <400> SEQUENCE: 19aagcatatcg ataaaaataa tgttaattgt gcag        #                  #        34 <210> SEQ ID NO 20 <211> LENGTH: 18 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial  #Sequence:Synthetic      Oligonucleotide <400> SEQUENCE: 20gccgagtcgt tctgaggg              #                   #                  #  18 <210> SEQ ID NO 21 <211> LENGTH: 43 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial  #Sequence:Synthetic      Oligonucleotide <400> SEQUENCE: 21gatcgaatcg atatgctttt ttattcattg ttatttttat ttc     #                  # 43 <210> SEQ ID NO 22 <211> LENGTH: 31 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial  #Sequence:Synthetic      Oligonucleotide <400> SEQUENCE: 22ccctgggatc caacactaac gttatttaac a         #                  #          31 <210> SEQ ID NO 23 <211> LENGTH: 37 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial  #Sequence:Synthetic      Oligonucleotide <400> SEQUENCE: 23gccgcatcga tatgcttttt tattcattta tatttta       #                  #      37 <210> SEQ ID NO 24 <211> LENGTH: 36 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial  #Sequence:Synthetic      Oligonucleotide <400> SEQUENCE: 24gccggcacgt gccatgattg aacgctattc ccaatg       #                  #       36 <210> SEQ ID NO 25 <211> LENGTH: 30 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial  #Sequence:Synthetic      Oligonucleotide <400> SEQUENCE: 25gccgggatcc ccctctattg ttagattgac          #                  #           30 <210> SEQ ID NO 26 <211> LENGTH: 30 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial  #Sequence:Synthetic      Oligonucleotide <400> SEQUENCE: 26acaacaagcc ggcacgtgcc atggaacgct          #                  #           30 <210> SEQ ID NO 27 <211> LENGTH: 24 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial  #Sequence:Synthetic      Oligonucleotide <400> SEQUENCE: 27tccttcttta ggtaccgctc tctc           #                  #                24 <210> SEQ ID NO 28 <211> LENGTH: 33 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial  #Sequence:Synthetic      Oligonucleotide <400> SEQUENCE: 28gggcttccat ggaacgctat tcccaatgtt cag        #                  #         33 <210> SEQ ID NO 29 <211> LENGTH: 35 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial  #Sequence:Synthetic      Oligonucleotide <400> SEQUENCE: 29cactaaggta ccttaacaat atttataacg tgcac        #                  #       35 <210> SEQ ID NO 30 <211> LENGTH: 25 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial  #Sequence:Synthetic      Oligonucleotide <400> SEQUENCE: 30ccattaagct tggtttcaaa caatc           #                  #               25 <210> SEQ ID NO 31 <211> LENGTH: 20 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial  #Sequence:Synthetic      Oligonucleotide <400> SEQUENCE: 31gtggcttcca ttgttggatc             #                  #                   # 20 <210> SEQ ID NO 32 <211> LENGTH: 20<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial  #Sequence:Synthetic      Oligonucleotide <400> SEQUENCE: 32caatgccgag tgtgcaaaga             #                  #                   # 20 <210> SEQ ID NO 33 <211> LENGTH: 20<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial  #Sequence:Synthetic      Oligonucleotide <400> SEQUENCE: 33caatgccgag tgtgcaaagg             #                  #                   # 20 <210> SEQ ID NO 34 <211> LENGTH: 67<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial  #Sequence:Synthetic      Oligonucleotide <400> SEQUENCE: 34aggcgagtcg acgtttcaaa caatcatcaa gtcaggttga agatgcatct ca#ggtaaaga     60 tagaagc                  #                  #                   #          67 <210> SEQ ID NO 35 <211> LENGTH: 67<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial  #Sequence:Synthetic      Oligonucleotide <400> SEQUENCE: 35aggcgagtcg acgtttcaaa caatcatcaa gtcaggttga agatgcatat ca#ggtaaaga     60 tagaagc                  #                  #                   #          67 <210> SEQ ID NO 36 <211> LENGTH: 67<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial  #Sequence:Synthetic      Oligonucleotide <400> SEQUENCE: 36aggcgagtcg acgtttcaaa caatcatcaa gtcaggttga agatgcatcc ta#ggtaaaga     60 tagaagc                  #                  #                   #          67 <210> SEQ ID NO 37 <211> LENGTH: 67<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial  #Sequence:Synthetic      Oligonucleotide <400> SEQUENCE: 37aggcgagtcg acgtttcaaa caatcatcaa gtcaggttga agatgcatct ta#ggtaaaga     60 tagaagc                  #                  #                   #          67 <210> SEQ ID NO 38 <211> LENGTH: 67<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial  #Sequence:Synthetic      Oligonucleotide <400> SEQUENCE: 38aggcgagtcg acgtttcaaa caatcatcaa gtcaggttga agatgcatat ta#ggtaaaga     60 tagaagc                  #                  #                   #          67 <210> SEQ ID NO 39 <211> LENGTH: 67<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial  #Sequence:Synthetic      Oligonucleotide <400> SEQUENCE: 39aggcgagtcg acgtttcaaa caatcatcaa gtcaggttga agatgcatca ta#ggtaaaga     60 tagaagc                  #                  #                   #          67 <210> SEQ ID NO 40 <211> LENGTH: 67<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial  #Sequence:Synthetic      Oligonucleotide <400> SEQUENCE: 40aggcgagtcg acgtttcaaa caatcatcaa gtcaggttga agatgcataa ta#ggtaaaga     60 tagaagc                  #                  #                   #          67 <210> SEQ ID NO 41 <211> LENGTH: 67<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial  #Sequence:Synthetic      Oligonucleotide <400> SEQUENCE: 41aggcgagtcg acgtttcaaa caatcatcaa gtcaggttga agatgcattg ta#ggtaaaga     60 tagaagc                  #                  #                   #          67 <210> SEQ ID NO 42 <211> LENGTH: 67<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial  #Sequence:Synthetic      Oligonucleotide <400> SEQUENCE: 42aggcgagtcg acgtttcaaa caatcatcaa gtcaggttga agatgcatcc aa#ggtaaaga     60 tagaagc                  #                  #                   #          67 <210> SEQ ID NO 43 <211> LENGTH: 67<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial  #Sequence:Synthetic      Oligonucleotide <400> SEQUENCE: 43aggcgagtcg acgtttcaaa caatcatcaa gtcaggttga agatgcatac aa#ggtaaaga     60 tagaagc                  #                  #                   #          67 <210> SEQ ID NO 44 <211> LENGTH: 67<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial  #Sequence:Synthetic      Oligonucleotide <400> SEQUENCE: 44aggcgagtcg acgtttcaaa caatcatcaa gtcaggttga agatgcatgt aa#ggtaaaga     60 tagaagc                  #                  #                   #          67 <210> SEQ ID NO 45 <211> LENGTH: 67<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial  #Sequence:Synthetic      Oligonucleotide <400> SEQUENCE: 45aggcgagtcg acgtttcaaa caatcatcaa gtcaggttga agatgcatga aa#ggtaaaga     60 tagaagc                  #                  #                   #          67 <210> SEQ ID NO 46 <211> LENGTH: 67<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial  #Sequence:Synthetic      Oligonucleotide <400> SEQUENCE: 46aggcgagtcg acgtttcaaa caatcatcaa gtcaggttga agatgcattt ga#ggtaaaga     60 tagaagc                  #                  #                   #          67 <210> SEQ ID NO 47 <211> LENGTH: 67<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial  #Sequence:Synthetic      Oligonucleotide <400> SEQUENCE: 47aggcgagtcg acgtttcaaa caatcatcaa gtcaggttga agatgcatgt ga#ggtaaaga     60 tagaagc                  #                  #                   #          67 <210> SEQ ID NO 48 <211> LENGTH: 67<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial  #Sequence:Synthetic      Oligonucleotide <400> SEQUENCE: 48aggcgagtcg acgtttcaaa caatcatcaa gtcaggttga agatgcatca ga#ggtaaaga     60 tagaagc                  #                  #                   #          67 <210> SEQ ID NO 49 <211> LENGTH: 67<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial  #Sequence:Synthetic      Oligonucleotide <400> SEQUENCE: 49aggcgagtcg acgtttcaaa caatcatcaa gtcaggttga agatgcatcg ga#ggtaaaga     60 tagaagc                  #                  #                   #          67 <210> SEQ ID NO 50 <211> LENGTH: 67<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial  #Sequence:Synthetic      Oligonucleotide <400> SEQUENCE: 50aggcgagtcg acgtttcaaa caatcatcaa gtcaggttga agatgcatgc ca#ggtaaaga     60 tagaagc                  #                  #                   #          67 <210> SEQ ID NO 51 <211> LENGTH: 67<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial  #Sequence:Synthetic      Oligonucleotide <400> SEQUENCE: 51aggcgagtcg acgtttcaaa caatcatcaa gtcaggttga agatgcataa ca#ggtaaaga     60 tagaagc                  #                  #                   #          67 <210> SEQ ID NO 52 <211> LENGTH: 33<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial  #Sequence:Synthetic      Oligonucleotide <400> SEQUENCE: 52aatgcagaat tcacaacacc actagcaggt ttg        #                  #         33 <210> SEQ ID NO 53 <211> LENGTH: 36 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial  #Sequence:Synthetic      Oligonucleotide <400> SEQUENCE: 53aggcgagtcg acgtttcaaa caatcatcaa gtcagg       #                  #       36 <210> SEQ ID NO 54 <211> LENGTH: 32 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial  #Sequence:Synthetic      Oligonucleotide <400> SEQUENCE: 54gccgaccggt agatctgaca aagcagcatt ag        #                  #          32 <210> SEQ ID NO 55 <211> LENGTH: 29 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial  #Sequence:Synthetic      Oligonucleotide <400> SEQUENCE: 55cggcggtacc gctctctctc tccccttgc          #                  #            29 <210> SEQ ID NO 56 <211> LENGTH: 5757 <212> TYPE: DNA<213> ORGANISM: Oryza sativa <400> SEQUENCE: 56gatgccgtcg tatactcgta tgctgctgcc ttctatcgat cgatcaaact cc#cgagtccc     60gagtccgagt cgcgtacgcg tgccggcgtg cggcgccgtg tgcgtccgca gt#tcgcaggc    120cgcacgctcg gctgggctgg ctctagtcct actgggcttc tcgcaggtgg gg#gcccctgg    180aaattggggg ccccctgcga tcgcccagct cgctctccgc gatcgacggg cc#tgggtagt    240tagcaccttg atcctggtta ccactacctc tcgagtagat ggcatctact cg#agaggtag    300tggcagccaa gaattaacaa cataggccgg tgatgacgag ggagacagca cc#atcggtga    360ccatgggaac aagggagagg gcattaatgg aagtgttgct aacccaaagc ta#ctactatg    420aagataagta ctgtaggatt agtactctca actcagtggc atgcttttat cc#aaaaggct    480acaatcaaga gttgcttcca gggcaaatgc ccttagttat gctttttttt tt#taatcccc    540aatgggctct cctctgtaga attttcttcc aactttatcc tctttggagt tt#ggcctaac    600ctaacatcta attatatagt taccatcgta caacgttact ctcaagcgag aa#ccaagtcg    660tcatagttcc aaagtaggaa aagttattcc attaattagc tactcggtcc at#gcaaagat    720cgtaagcata tttattttat catcgagacc aagaaaaaaa attaactcat tc#gatttgta    780ctcccatgta acaaactttt tcatatgcat gcaaaagggt tggagaatgc at#ggcagctc    840aagtttcggt caacaattta atcatacacg gtaaaacgaa aagagactct ct#tcatttaa    900ggctcggatg catggagact atatgaataa gctcatctca tttggaaaaa aa#taatcaaa    960acaatgaaat atgcttaata catttggatt tggatggagt atctccctat ga#cgaaagag   1020acgagggatt gcctacttct ttccacatca acctttgaag gctatggcag ag#ctacattt   1080gcagcgcagc ctctctgcat gatttcttac tagataggat taagtttcaa ga#gttaattt   1140cttgtactct tgggcgatcc atgctgtcaa atcagcaact aacgaggcat aa#tctcgatc   1200actagctctg atctgatctt cagctagcgc cggcgagctg cagaggtctc ca#tccatggc   1260gccggcggtc attgcatcgc agggtgtcat catgcggtcc ctgacgagca ag#ctcgactc   1320gctgctgctg cagccgccgg agccgccgcc gcctgcgcaa ccgtcgtcgc tg#cggaaggg   1380ggagaggaag aagatcctcc tcctcagagg cgatctccga cacctgctag at#gactacta   1440cctcctcgtg gagccgccgt cagacaccgc gccaccgcca gactcgacgg cg#gcgtgctg   1500ggctaaggag gttcgcgagc tctcctacga cgtcgacgac ttcctcgacg ag#ctaacgac   1560ccagctcctc caccaccgcg gcggcggcga tggcagtagc actgctggtg cc#aagaagat   1620gatcagcagc atgatcgcgc ggcttcgagg ggagcttaac cggcggcggt gg#atcgccga   1680cgaggtcacc ctgttcaggg cccgcgtgaa ggaggccatt cgccgccacg ag#agctacca   1740tcttggcagg cgcacctcga gctcgaggcc gagagaagaa gacgacgacg ac#gatcgcga   1800ggactccgcc ggcaacgaac gccgccggtt tctgtcgctg acgttcggga tg#gacgacgc   1860tgctgtgcac ggccagctcg ttggtaggga tatttcgatg caaaagctcg tc#cggtggct   1920ggccgacggc gagccgaagc tcaaggtggc ttccattgtt ggatccggag gt#gttggcaa   1980gacgacgctg gccacagaat tctatcgtct gcatggccgg cggttggatg cg#ccgttcga   2040ctgccgggct ttcgtgcgga cgccccggaa gcctgacatg acgaagatcc tc#accgacat   2100gctgtcacag ctgcggccac aacatcagca tcagtcttcg gatgtttggg ag#gttgatcg   2160actccttgaa actatccgga cgcatttgca agataaaagg taattcatgt ct#acatctat   2220ctctagtatt tttttcatga atttacaaac tattttctca aatttcccct tt#tttatcct   2280tcatatagta attaagttag taactataca tatgaattta atttactcga ca#atgccaat   2340aatattttaa attactttta tagtgtcctc tattattaaa tacaattcga tc#gagccatg   2400aatcatactg gtaatctaaa caattattat acccacctca ttctactaac ag#caattgag   2460ttgttaatat aaatagatat ggtctattgc atgaaaaaat aaaggtaaag tg#tttacatt   2520atttctacta ctagtagcag aactacaaag ttgtttgtat ttttaattat ta#aatgtaaa   2580tgaaagtaca tgtttcatac ccttgttatt tttttaggcg actaaactac ta#gtacatta   2640tttctactac tagtagaaca actacaaagt tgtttgtatt tttaattact aa#atgtaaat   2700gaaagtacat gcttcatacc cttgttataa tattttagac taaactacca ca#atggcatg   2760ttaacttata gcagtaagtt gtaatttatt ttcttttcat tttctcagtt gt#tacaagaa   2820ctttttttta cttaataaaa aatagtcgta agggcctccc ttgttcggtc aa#aaaagaat   2880tagactaaac tcaatcgtat ttgtacataa acaaaattta aaatataact aa#aacgaaac   2940agtgataaaa atggtgcaat ttaactgctg cctcttgttt taatgtctga ca#atgttgat   3000tttgtatata tgtttggcca tttattttat tcaaattttt tacaaatatg aa#aaatataa   3060tatgtgctta aattactttt aatgataaaa taaatttaag aaaatgataa tt#atattttt   3120aataagatgg atgatctaac atatatatgt ccaaaagttt gacatcaaac at#taaaaaca   3180agagagagta tttgcttagg agtacgtgtc tttttccatg ccttagaaca gg#tacaatag   3240caggttataa gctaggccaa acatatttta aagagatata ggaagagaga ga#agagcagc   3300agcctacaga tctgtagcca gctgcagcac ggactctaag acgtaatgtg tg#tatgacag   3360gtaggaccaa gtattaatag tatagtaagc aactattgta tgaattggct at#ttggctct   3420agatgatttg gagctagtag tcggctatac tattaaactt gctcttagat at#gtggttaa   3480atttaatttc tgaaatttac aagatataga acatgtcttc accatgtttg ca#aagttgat   3540aaaatgaaat tgcactcaac tttttaaatt cagcatgcta tcccacgtat ag#ctttttaa   3600atatgattta ttttttcgta cattagaagt ttaatgttta cgaagtacta aa#tgttctgc   3660aggtacttca tcataattga agatttatgg gcttcatcaa tgtgggatat tg#ttagccgt   3720ggtttgcctg ataataatag ttgcagtaga atactaataa caacagaaat tg#aacctgta   3780gctttggcat gctgtggata taactcagag cacattatta agattgatcc ac#tgggtgat   3840gatgtctcaa gtcaattgtt tttcagtgga gttgttggcc aaggaaatga at#ttcctgga   3900catcttactg aagtttctca tgacatgata aaaaaatgtg gtggcttgcc ac#tagcaata   3960actataacag ccagacattt taaaagccag ctgttagatg gaatgcagca at#ggaatcac   4020atacaaaaat cattgactac ttccaatttg aagaaaaatc ctactttgca gg#ggatgagg   4080caagtactca accttattta caataatctt cctcattgtt tgaaagcatg tc#tgttatac   4140cttagcatct acaaagagga ctacataatt aggaaggcca acttggtgag gc#aatggatg   4200gctgaaggtt tcatcaattc catagaaaat aaagtcatgg aagaagttgc ag#ggaactat   4260tttgatgaac ttgttggtag gggcctggtc caaccagtag atgttaactg ca#aaaatgag   4320gtattgtcat gtgtagtgca ccacatggta ttaaatttca tcaggtgtaa gt#caatagag   4380gagaatttca gcattacatt ggatcattct cagacgacag taagacatgc tg#acaaggtt   4440cgccgactct cgcttcactt cagcaatgca catgatacaa caccactagc ag#gtttgaga   4500ctctcacaag ttcgatcgat ggcatttttc ggacaagtca agtgtatgcc tt#ccattgca   4560gattataggc ttcttcgagt tctgattctt tgtttttggg ctgatcaaga ga#aaacaagc   4620tatgacctca caagcatttt tgaactgtta caactgagat atctgaagat aa#caggtaat   4680atcacagtta aacttccaga gaagatccaa ggactacaac acttgcagac ac#tggaagca   4740gatgcaagag caactgctgt cctattggat attgttcata cacagtgttt gt#tgcacctt   4800cgtcttgtac tacttgatct gctccctcac tgtcacaggt acatcttcac ca#gcatcccc   4860aaatggactg gaaagctcaa caatctccgc attttaaaca ttgcagtcat gc#aaatttcc   4920caggatgacc ttgacactct caaaggactg ggatctctca ctgctctttc gc#tgcttgtt   4980cgaacagcgc ctgcgcaaag aatcgtcgct gcgaatgagg ggttcgggtc tc#tcaagtac   5040ttcatgtttg tctgtacagc accatgcatg acttttgtgg aaggagcaat gc#cgagtgtg   5100caaaggttaa atctaaggtt caatgccaac gagttcaagc agtatgactc ta#aggagaca   5160gggttggaac acttggtcgc ccttgcagag atctctgcaa gaattggggg ca#ctgatgat   5220gatgaatcaa acaaaactga agtggagtct gccttgagga ctgcaattcg ca#agcatccg   5280acgccgagca ctcttatggt tgatatacaa tgggtggatt ggatctttgg tg#ctgaaggg   5340agagacttgg atgaagattt ggcacaacaa gatgatcacg ggtatggatt tt#tcattcta   5400ttcccaggtt acaacttaca aggattattg agcttctttc tttctctgcc gt#ggcttcta   5460tctttacctg ctatgcatct tcaacctgac ttgatgattg tttgaaacca at#tttaatgg   5520aagttaaatg ttattgttgt gaccctgaat caggttttgt atgctaccgg aa#tcctcttc   5580acgtcttcag agtagaggta attttgtttc ttgtcatttt acagttacag tt#catcttct   5640taaggaatta atgggaggtc ctatattttc tcgggtacct agaaggtgtg gt#tcctagtc   5700ttacctcctt aagaaccgta gaatgttggc cctaacactt ggatcaaaga ac#gaaag      5757 <210> SEQ ID NO 57 <211> LENGTH: 5222 <212> TYPE: DNA<213> ORGANISM: Oryza sativa <400> SEQUENCE: 57gaattctaag caaaactctc ctccaatgct ccctttcttt ttcaagttct cg#ttgggcaa     60ctttatcaat tgtttgattc ttttgcatcc taagacgcaa ttcataccat gt#ggacatat    120ttgtgacatg ctctctacta attcatgctc tttaagtcta ttagcaagat ga#ttccagtc    180attgacaccg tcatttgtta gctgacttct cacaagcccc tttctcaata at#ttgcaaca    240aaaacaaaat actttgtcaa gctctttgct gtaaacaagc cattccctgt ca#cacttctc    300tccatttgag agaactttag tgtatgatga tgcaagaaac cttctagaca aa#ttgtctct    360gggaccatgc tcaatagaca aatctctttt agggcccttt tgcaataaaa ta#tcaatcat    420tttaggatca agtccatccc aagttcttgg atcaaacata tcaggtcgaa aa#gaaacatt    480atcgtcggcg tcgtcagcaa tattttcctc attaccttca ctggcaagat ca#cggccttc    540atcggcaaga tcatggccaa tgtctccatc aacattttcc tctactgcgt ca#tcactttc    600ggcaatatta gcatcaacct ctgccatatc atcactaata tcgtcttcaa ta#ttagcatt    660tggagtttct ctcaaaaaaa atttatcaag agcacccttt tgagattgag ct#actgcttc    720tagtcttctt ctcttctggc gtttttcagc gccagaatca tacttcctat tt#ctagagga    780catgatcgct tcacttgatg aattgaggat tgaccgaccg aactgctata gt#actgtatt    840ctttctgttt aataaaaaac taaagtaact tcagtgattt taatcaaaca tg#aactgatc    900aattaaattt aatttaattt acattgtaca tttgtactca caaagtcaca at#ggagaaca    960ggagaagccg agaaggtccg acggcagcgg cggcgtggcg tcggcggcgg cg#gtggcgcg   1020gacggcagcg gcggcacggc gtcggccgga cggcagcggc gttcggctcg gc#tgcctgga   1080ttggatggcg aggcgacgag acgacgaggc ggcgagagcg ctaggagcct ag#ggctgcga   1140gtcgtgcgat gcgaggacag aaaccgaagc gatgccgtcg tatactcgta tg#ctgctgcc   1200ttctatcgat cgatcaaact cccgagtccc gagtccgagt cgcgtacgcg tg#ccggcgtg   1260cggcgccgtg tgcgtccgca gttcgcaggc cgcacgctcg gctgggctgg ct#ctagtcct   1320actgggcttc tcgcaggtgg gggcccctgg aaattggggg ccccctgcga tc#gcccagct   1380cgctctccgc gatcgacggg cctgggtagt tagcaccttg atcctggtta cc#actacctc   1440tcgagtagat ggcatctact cgagaggtag tggcagccaa gaattaacaa ca#taggccgg   1500tgatgacgag ggagacagca ccatcggtga ccatgggaac aagggagagg gc#attaatgg   1560aagtgttgct aacccaaagc tactactatg aagataagta ctgtaggatt ag#tactctca   1620actcagtggc atgcttttat ccaaaaggct acaatcaaga gttgcttcca gg#gcaaatgc   1680ccttagttat gctttttttt tttaatcccc aatgggctct cctctgtaga at#tttcttcc   1740aactttatcc tctttggagt ttggcctaac ctaacatcta attatatagt ta#ccatcgta   1800caacgttact ctcaagcgag aaccaagtcg tcatagttcc aaagtaggaa aa#gttattcc   1860attaattagc tactcggtcc atgcaaagat cgtaagcata tttattttat ca#tcgagacc   1920aagaaaaaaa attaactcat tcgatttgta ctcccatgta acaaactttt tc#atatgcat   1980gcaaaagggt tggagaatgc atggcagctc aagtttcggt caacaattta at#catacacg   2040gtaaaacgaa aagagactct cttcatttaa ggctcggatg catggagact at#atgaataa   2100gctcatctca tttggaaaaa aataatcaaa acaatgaaat atgcttaata ca#tttggatt   2160tggatggagt atctccctat gacgaaagag acgagggatt gcctacttct tt#ccacatca   2220acctttgaag gctatggcag agctacattt gcagcgcagc ctctctgcat ga#tttcttac   2280tagataggat taagtttcaa gagttaattt cttgtactct tgggcgatcc at#gctgtcaa   2340atcagcaact aacgaggcat aatctcgatc actagctctg atctgatctt ca#gctagcgc   2400cggcgagctg cagaggtctc catccatggc gccggcggtc attgcatcgc ag#ggtgtcat   2460catgcggtcc ctgacgagca agctcgactc gctgctgctg cagccgccgg ag#ccgccgcc   2520gcctgcgcaa ccgtcgtcgc tgcggaaggg ggagaggaag aagatcctcc tc#ctcagagg   2580cgatctccga cacctgctag atgactacta cctcctcgtg gagccgccgt ca#gacaccgc   2640gccaccgcca gactcgacgg cggcgtgctg ggctaaggag gttcgcgagc tc#tcctacga   2700cgtcgacgac ttcctcgacg agctaacgac ccagctcctc caccaccgcg gc#ggcggcga   2760tggcagtagc actgctggtg ccaagaagat gatcagcagc atgatcgcgc gg#cttcgagg   2820ggagcttaac cggcggcggt ggatcgccga cgaggtcacc ctgttcaggg cc#cgcgtgaa   2880ggaggccatt cgccgccacg agagctacca tcttggcagg cgcacctcga gc#tcgaggcc   2940gagagaagaa gacgacgacg acgatcgcga ggactccgcc ggcaacgaac gc#cgccggtt   3000tctgtcgctg acgttcggga tggacgacgc tgctgtgcac ggccagctcg tt#ggtaggga   3060tatttcgatg caaaagctcg tccggtggct ggccgacggc gagccgaagc tc#aaggtggc   3120ttccattgtt ggatccggag gtgttggcaa gacgacgctg gccacagaat tc#tatcgtct   3180gcatggccgg cggttggatg cgccgttcga ctgccgggct ttcgtgcgga cg#ccccggaa   3240gcctgacatg acgaagatcc tcaccgacat gctgtcacag ctgcggccac aa#catcagca   3300tcagtcttcg gatgtttggg aggttgatcg actccttgaa actatccgga cg#catttgca   3360agataaaagg tacttcatca taattgaaga tttatgggct tcatcaatgt gg#gatattgt   3420tagccgtggt ttgcctgata ataatagttg cagtagaata ctaataacaa ca#gaaattga   3480acctgtagct ttggcatgct gtggatataa ctcagagcac attattaaga tt#gatccact   3540gggtgatgat gtctcaagtc aattgttttt cagtggagtt gttggccaag ga#aatgaatt   3600tcctggacat cttactgaag tttctcatga catgataaaa aaatgtggtg gc#ttgccact   3660agcaataact ataacagcca gacattttaa aagccagctg ttagatggaa tg#cagcaatg   3720gaatcacata caaaaatcat tgactacttc caatttgaag aaaaatccta ct#ttgcaggg   3780gatgaggcaa gtactcaacc ttatttacaa taatcttcct cattgtttga aa#gcatgtct   3840gttatacctt agcatctaca aagaggacta cataattagg aaggccaact tg#gtgaggca   3900atggatggct gaaggtttca tcaattccat agaaaataaa gtcatggaag aa#gttgcagg   3960gaactatttt gatgaacttg ttggtagggg cctggtccaa ccagtagatg tt#aactgcaa   4020aaatgaggta ttgtcatgtg tagtgcacca catggtatta aatttcatca gg#tgtaagtc   4080aatagaggag aatttcagca ttacattgga tcattctcag acgacagtaa ga#catgctga   4140caaggttcgc cgactctcgc ttcacttcag caatgcacat gatacaacac ca#ctagcagg   4200tttgagactc tcacaagttc gatcgatggc atttttcgga caagtcaagt gt#atgccttc   4260cattgcagat tataggcttt ttcgagttct gattctttgt ttttgggctg at#caagagaa   4320aacaagctat gacctcacaa gcatttttga actgttacaa ctgagatatc tg#aagataac   4380aggtaatatc acagttaaac ttccagagaa gatccaagga ctacaacact tg#cagacact   4440ggaagcagat gcaagagcaa ctgctgtcct attggatatt gttcatacac ag#tgtttgtt   4500gcaccttcgt cttgtactac ttgatctgct ccctcactgt cacaggtaca tc#ttcaccag   4560catccccaaa tggactggaa agctcaacaa tctccgcatt ttaaacattg ca#gtcatgca   4620aatttcccag gatgaccttg acactctcaa aggactggga tctctcactg ct#ctttcgct   4680gcttgttcga acagcgcctg cgcaaagaat cgtcgctgcg aatgaggggt tc#gggtctct   4740caagtacttc atgtttgtct gtacagcacc atgcatgact tttgtggaag ga#gcaatgcc   4800gagtgtgcaa aggttaaatc taaggttcaa tgccaacgag ttcaagcagt at#gactctaa   4860ggagacaggg ttggaacact tggtcgccct tgcagagatc tctgcaagaa tt#gggggcac   4920tgatgatgat gaatcaaaca aaactgaagt ggagtctgcc ttgaggactg ca#attcgcaa   4980gcatccgacg ccgagcactc ttatggttga tatacaatgg gtggattgga tc#tttggtgc   5040tgaagggaga gacttggatg aagatttggc acaacaagat gatcacgggt at#ggattttt   5100cattctattc ccaggttaca acttacaagg attattgagc ttctttcttt ct#ctgccgtg   5160gcttctatct ttacctgcta tgcatcttca acctgacttg atgattgttt ga#aaccaagc   5220 tt                   #                  #                   #            5222 <210> SEQ ID NO 58<211> LENGTH: 928 <212> TYPE: PRT <213> ORGANISM: Oryza sativa<400> SEQUENCE: 58 Met Ala Pro Ala Val Ile Ala Ser Gln Gly Va#l Ile Met Arg Ser Leu 1               5    #                10  #                15 Thr Ser Lys Leu Asp Ser Leu Leu Leu Gln Pr#o Pro Glu Pro Pro Pro              20      #             25     #             30 Pro Ala Gln Pro Ser Ser Leu Arg Lys Gly Gl#u Arg Lys Lys Ile Leu          35          #         40         #         45 Leu Leu Arg Gly Asp Leu Arg His Leu Leu As#p Asp Tyr Tyr Leu Leu      50              #     55             #     60 Val Glu Pro Pro Ser Asp Thr Ala Pro Pro Pr#o Asp Ser Thr Ala Ala  65                  # 70                 # 75                  # 80 Cys Trp Ala Lys Glu Val Arg Glu Leu Ser Ty#r Asp Val Asp Asp Phe                  85  #                 90 #                 95 Leu Asp Glu Leu Thr Thr Gln Leu Leu His Hi#s Arg Gly Gly Gly Asp             100       #           105      #           110 Gly Ser Ser Thr Ala Gly Ala Lys Lys Met Il#e Ser Ser Met Ile Ala         115           #       120          #       125 Arg Leu Arg Gly Glu Leu Asn Arg Arg Arg Tr#p Ile Ala Asp Glu Val     130               #   135              #   140 Thr Leu Phe Arg Ala Arg Val Lys Glu Ala Il#e Arg Arg His Glu Ser 145                 1 #50                 1#55                 1 #60 Tyr His Leu Gly Arg Arg Thr Ser Ser Ser Ar#g Pro Arg Glu Glu Asp                 165   #               170  #               175 Asp Asp Asp Asp Arg Glu Asp Ser Ala Gly As#n Glu Arg Arg Arg Phe             180       #           185      #           190 Leu Ser Leu Thr Phe Gly Met Asp Asp Ala Al#a Val His Gly Gln Leu         195           #       200          #       205 Val Gly Arg Asp Ile Ser Met Gln Lys Leu Va#l Arg Trp Leu Ala Asp     210               #   215              #   220 Gly Glu Pro Lys Leu Lys Val Ala Ser Ile Va#l Gly Ser Gly Gly Val 225                 2 #30                 2#35                 2 #40 Gly Lys Thr Thr Leu Ala Thr Glu Phe Tyr Ar#g Leu His Gly Arg Arg                 245   #               250  #               255 Leu Asp Ala Pro Phe Asp Cys Arg Ala Phe Va#l Arg Thr Pro Arg Lys             260       #           265      #           270 Pro Asp Met Thr Lys Ile Leu Thr Asp Met Le#u Ser Gln Leu Arg Pro         275           #       280          #       285 Gln His Gln His Gln Ser Ser Asp Val Trp Gl#u Val Asp Arg Leu Leu     290               #   295              #   300 Glu Thr Ile Arg Thr His Leu Gln Asp Lys Ar#g Tyr Phe Ile Ile Ile 305                 3 #10                 3#15                 3 #20 Glu Asp Leu Trp Ala Ser Ser Met Trp Asp Il#e Val Ser Arg Gly Leu                 325   #               330  #               335 Pro Asp Asn Asn Ser Cys Ser Arg Ile Leu Il#e Thr Thr Glu Ile Glu             340       #           345      #           350 Pro Val Ala Leu Ala Cys Cys Gly Tyr Asn Se#r Glu His Ile Ile Lys         355           #       360          #       365 Ile Asp Pro Leu Gly Asp Asp Val Ser Ser Gl#n Leu Phe Phe Ser Gly     370               #   375              #   380 Val Val Gly Gln Gly Asn Glu Phe Pro Gly Hi#s Leu Thr Glu Val Ser 385                 3 #90                 3#95                 4 #00 His Asp Met Ile Lys Lys Cys Gly Gly Leu Pr#o Leu Ala Ile Thr Ile                 405   #               410  #               415 Thr Ala Arg His Phe Lys Ser Gln Leu Leu As#p Gly Met Gln Gln Trp             420       #           425      #           430 Asn His Ile Gln Lys Ser Leu Thr Thr Ser As#n Leu Lys Lys Asn Pro         435           #       440          #       445 Thr Leu Gln Gly Met Arg Gln Val Leu Asn Le#u Ile Tyr Asn Asn Leu     450               #   455              #   460 Pro His Cys Leu Lys Ala Cys Leu Leu Tyr Le#u Ser Ile Tyr Lys Glu 465                 4 #70                 4#75                 4 #80 Asp Tyr Ile Ile Arg Lys Ala Asn Leu Val Ar#g Gln Trp Met Ala Glu                 485   #               490  #               495 Gly Phe Ile Asn Ser Ile Glu Asn Lys Val Me#t Glu Glu Val Ala Gly             500       #           505      #           510 Asn Tyr Phe Asp Glu Leu Val Gly Arg Gly Le#u Val Gln Pro Val Asp         515           #       520          #       525 Val Asn Cys Lys Asn Glu Val Leu Ser Cys Va#l Val His His Met Val     530               #   535              #   540 Leu Asn Phe Ile Arg Cys Lys Ser Ile Glu Gl#u Asn Phe Ser Ile Thr 545                 5 #50                 5#55                 5 #60 Leu Asp His Ser Gln Thr Thr Val Arg His Al#a Asp Lys Val Arg Arg                 565   #               570  #               575 Leu Ser Leu His Phe Ser Asn Ala His Asp Th#r Thr Pro Leu Ala Gly             580       #           585      #           590 Leu Arg Leu Ser Gln Val Arg Ser Met Ala Ph#e Phe Gly Gln Val Lys         595           #       600          #       605 Cys Met Pro Ser Ile Ala Asp Tyr Arg Leu Le#u Arg Val Leu Ile Leu     610               #   615              #   620 Cys Phe Trp Ala Asp Gln Glu Lys Thr Ser Ty#r Asp Leu Thr Ser Ile 625                 6 #30                 6#35                 6 #40 Phe Glu Leu Leu Gln Leu Arg Tyr Leu Lys Il#e Thr Gly Asn Ile Thr                 645   #               650  #               655 Val Lys Leu Pro Glu Lys Ile Gln Gly Leu Gl#n His Leu Gln Thr Leu             660       #           665      #           670 Glu Ala Asp Ala Arg Ala Thr Ala Val Leu Le#u Asp Ile Val His Thr         675           #       680          #       685 Gln Cys Leu Leu His Leu Arg Leu Val Leu Le#u Asp Leu Leu Pro His     690               #   695              #   700 Cys His Arg Tyr Ile Phe Thr Ser Ile Pro Ly#s Trp Thr Gly Lys Leu 705                 7 #10                 7#15                 7 #20 Asn Asn Leu Arg Ile Leu Asn Ile Ala Val Me#t Gln Ile Ser Gln Asp                 725   #               730  #               735 Asp Leu Asp Thr Leu Lys Gly Leu Gly Ser Le#u Thr Ala Leu Ser Leu             740       #           745      #           750 Leu Val Arg Thr Ala Pro Ala Gln Arg Ile Va#l Ala Ala Asn Glu Gly         755           #       760          #       765 Phe Gly Ser Leu Lys Tyr Phe Met Phe Val Cy#s Thr Ala Pro Cys Met     770               #   775              #   780 Thr Phe Val Glu Gly Ala Met Pro Ser Val Gl#n Arg Leu Asn Leu Arg 785                 7 #90                 7#95                 8 #00 Phe Asn Ala Asn Glu Phe Lys Gln Tyr Asp Se#r Lys Glu Thr Gly Leu                 805   #               810  #               815 Glu His Leu Val Ala Leu Ala Glu Ile Ser Al#a Arg Ile Gly Gly Thr             820       #           825      #           830 Asp Asp Asp Glu Ser Asn Lys Thr Glu Val Gl#u Ser Ala Leu Arg Thr         835           #       840          #       845 Ala Ile Arg Lys His Pro Thr Pro Ser Thr Le#u Met Val Asp Ile Gln     850               #   855              #   860 Trp Val Asp Trp Ile Phe Gly Ala Glu Gly Ar#g Asp Leu Asp Glu Asp 865                 8 #70                 8#75                 8 #80 Leu Ala Gln Gln Asp Asp His Gly Tyr Gly Ph#e Phe Ile Leu Phe Pro                 885   #               890  #               895 Gly Tyr Asn Leu Gln Gly Leu Leu Ser Phe Ph#e Leu Ser Leu Pro Trp             900       #           905      #           910 Leu Leu Ser Leu Pro Ala Met His Leu Gln Pr#o Asp Leu Met Ile Val         915           #       920          #       925 <210> SEQ ID NO 59 <211> LENGTH: 881 <212> TYPE: DNA<213> ORGANISM: Magnaporthe grisea <400> SEQUENCE: 59atgctttttt attcattatt tttttttcac accgttgcga tttcggcctt ca#ccaacatt     60ggcacctttt cacacccagt ttacgattac aatccaattc caaaccatat cc#acggagat    120ttaaaaaggc gggcttatat tgaacgctat tcccaatgtt cagattcgca gg#cctccgaa    180attcgtgccg cgctaaaaag gtaaattgaa actctttaaa acaaattcgg aa#aacaatgt    240taaatatttt gtttagttgc gccgagctcg cctcgtgggg ctatcacgcc gt#taaaagta    300acaatcggtt atttaaatta atctttaaaa ctgacagcac agatattcaa aa#ctgggttc    360aaaataattt taacgaaatt tacaaggaat gtaacaggga cgcggacgaa at#ttctctaa    420cctgccacga taaaaatgtt tatacgtgcg tccgagaagg agttcataat tt#ggcgtatg    480cacttattaa cgaaaaagaa attgttatat gccctccttt cttcaacaac cc#cgtaaaca    540gcagggaaat tactgccggt aaccaagata caattatatt acatgaaatg gt#gcatataa    600ttttaagtaa gtttgctttt acaaattgat aaaacattta caaaagttta tt#tataaaaa    660ttttcaaaaa ctaaaaattc aaatttttat ttagaagagt ggaaagatta tg#gttgcgaa    720tgggatggga ttcacaagta agttgtcgaa aaacaaaatg ctgaatgttg tt#ttatattg    780ataaattcta attaatatta agattggata gtacagaaag tattaaaaac cc#cgacagtt    840 atgctatttt tgcacaatgt gcacgttata aatattgtta a    #                   #  881 <210> SEQ ID NO 60 <211> LENGTH: 5696<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial  #Sequence:Chimeric      Gene <400> SEQUENCE: 60gaattctaag caaaactctc ctccaatgct ccctttcttt ttcaagttct cg#ttgggcaa     60ctttatcaat tgtttgattc ttttgcatcc taagacgcaa ttcataccat gt#ggacatat    120ttgtgacatg ctctctacta attcatgctc tttaagtcta ttagcaagat ga#ttccagtc    180attgacaccg tcatttgtta gctgacttct cacaagcccc tttctcaata at#ttgcaaca    240aaaacaaaat actttgtcaa gctctttgct gtaaacaagc cattccctgt ca#cacttctc    300tccatttgag agaactttag tgtatgatga tgcaagaaac cttctagaca aa#ttgtctct    360gggaccatgc tcaatagaca aatctctttt agggcccttt tgcaataaaa ta#tcaatcat    420tttaggatca agtccatccc aagttcttgg atcaaacata tcaggtcgaa aa#gaaacatt    480atcgtcggcg tcgtcagcaa tattttcctc attaccttca ctggcaagat ca#cggccttc    540atcggcaaga tcatggccaa tgtctccatc aacattttcc tctactgcgt ca#tcactttc    600ggcaatatta gcatcaacct ctgccatatc atcactaata tcgtcttcaa ta#ttagcatt    660tggagtttct ctcaaaaaaa atttatcaag agcacccttt tgagattgag ct#actgcttc    720tagtcttctt ctcttctggc gtttttcagc gccagaatca tacttcctat tt#ctagagga    780catgatcgct tcacttgatg aattgaggat tgaccgaccg aactgctata gt#actgtatt    840ctttctgttt aataaaaaac taaagtaact tcagtgattt taatcaaaca tg#aactgatc    900aattaaattt aatttaattt acattgtaca tttgtactca caaagtcaca at#ggagaaca    960ggagaagccg agaaggtccg acggcagcgg cggcgtggcg tcggcggcgg cg#gtggcgcg   1020gacggcagcg gcggcacggc gtcggccgga cggcagcggc gttcggctcg gc#tgcctgga   1080ttggatggcg aggcgacgag acgacgaggc ggcgagagcg ctaggagcct ag#ggctgcga   1140gtcgtgcgat gcgaggacag aaaccgaagc gatgccgtcg tatactcgta tg#ctgctgcc   1200ttctatcgat cgatcaaact cccgagtccc gagtccgagt cgcgtacgcg tg#ccggcgtg   1260cggcgccgtg tgcgtccgca gttcgcaggc cgcacgctcg gctgggctgg ct#ctagtcct   1320actgggcttc tcgcaggtgg gggcccctgg aaattggggg ccccctgcga tc#gcccagct   1380cgctctccgc gatcgacggg cctgggtagt tagcaccttg atcctggtta cc#actacctc   1440tcgagtagat ggcatctact cgagaggtag tggcagccaa gaattaacaa ca#taggccgg   1500tgatgacgag ggagacagca ccatcggtga ccatgggaac aagggagagg gc#attaatgg   1560aagtgttgct aacccaaagc tactactatg aagataagta ctgtaggatt ag#tactctca   1620actcagtggc atgcttttat ccaaaaggct acaatcaaga gttgcttcca gg#gcaaatgc   1680ccttagttat gctttttttt tttaatcccc aatgggctct cctctgtaga at#tttcttcc   1740aactttatcc tctttggagt ttggcctaac ctaacatcta attatatagt ta#ccatcgta   1800caacgttact ctcaagcgag aaccaagtcg tcatagttcc aaagtaggaa aa#gttattcc   1860attaattagc tactcggtcc atgcaaagat cgtaagcata tttattttat ca#tcgagacc   1920aagaaaaaaa attaactcat tcgatttgta ctcccatgta acaaactttt tc#atatgcat   1980gcaaaagggt tggagaatgc atggcagctc aagtttcggt caacaattta at#catacacg   2040gtaaaacgaa aagagactct cttcatttaa ggctcggatg catggagact at#atgaataa   2100gctcatctca tttggaaaaa aataatcaaa acaatgaaat atgcttaata ca#tttggatt   2160tggatggagt atctccctat gacgaaagag acgagggatt gcctacttct tt#ccacatca   2220acctttgaag gctatggcag agctacattt gcagcgcagc ctctctgcat ga#tttcttac   2280tagataggat taagtttcaa gagttaattt cttgtactct tgggcgatcc at#gctgtcaa   2340atcagcaact aacgaggcat aatctcgatc actagctctg atctgatctt ca#gctagcgc   2400cggcgagctg cagaggtctc catccatggc gccggcggtc attgcatcgc ag#ggtgtcat   2460catgcggtcc ctgacgagca agctcgactc gctgctgctg cagccgccgg ag#ccgccgcc   2520gcctgcgcaa ccgtcgtcgc tgcggaaggg ggagaggaag aagatcctcc tc#ctcagagg   2580cgatctccga cacctgctag atgactacta cctcctcgtg gagccgccgt ca#gacaccgc   2640gccaccgcca gactcgacgg cggcgtgctg ggctaaggag gttcgcgagc tc#tcctacga   2700cgtcgacgac ttcctcgacg agctaacgac ccagctcctc caccaccgcg gc#ggcggcga   2760tggcagtagc actgctggtg ccaagaagat gatcagcagc atgatcgcgc gg#cttcgagg   2820ggagcttaac cggcggcggt ggatcgccga cgaggtcacc ctgttcaggg cc#cgcgtgaa   2880ggaggccatt cgccgccacg agagctacca tcttggcagg cgcacctcga gc#tcgaggcc   2940gagagaagaa gacgacgacg acgatcgcga ggactccgcc ggcaacgaac gc#cgccggtt   3000tctgtcgctg acgttcggga tggacgacgc tgctgtgcac ggccagctcg tt#ggtaggga   3060tatttcgatg caaaagctcg tccggtggct ggccgacggc gagccgaagc tc#aaggtggc   3120ttccattgtt ggatccggag gtgttggcaa gacgacgctg gccacagaat tc#tatcgtct   3180gcatggccgg cggttggatg cgccgttcga ctgccgggct ttcgtgcgga cg#ccccggaa   3240gcctgacatg acgaagatcc tcaccgacat gctgtcacag ctgcggccac aa#catcagca   3300tcagtcttcg gatgtttggg aggttgatcg actccttgaa actatccgga cg#catttgca   3360agataaaagg tacttcatca taattgaaga tttatgggct tcatcaatgt gg#gatattgt   3420tagccgtggt ttgcctgata ataatagttg cagtagaata ctaataacaa ca#gaaattga   3480acctgtagct ttggcatgct gtggatataa ctcagagcac attattaaga tt#gatccact   3540gggtgatgat gtctcaagtc aattgttttt cagtggagtt gttggccaag ga#aatgaatt   3600tcctggacat cttactgaag tttctcatga catgataaaa aaatgtggtg gc#ttgccact   3660agcaataact ataacagcca gacattttaa aagccagctg ttagatggaa tg#cagcaatg   3720gaatcacata caaaaatcat tgactacttc caatttgaag aaaaatccta ct#ttgcaggg   3780gatgaggcaa gtactcaacc ttatttacaa taatcttcct cattgtttga aa#gcatgtct   3840gttatacctt agcatctaca aagaggacta cataattagg aaggccaact tg#gtgaggca   3900atggatggct gaaggtttca tcaattccat agaaaataaa gtcatggaag aa#gttgcagg   3960gaactatttt gatgaacttg ttggtagggg cctggtccaa ccagtagatg tt#aactgcaa   4020aaatgaggta ttgtcatgtg tagtgcacca catggtatta aatttcatca gg#tgtaagtc   4080aatagaggag aatttcagca ttacattgga tcattctcag acgacagtaa ga#catgctga   4140caaggttcgc cgactctcgc ttcacttcag caatgcacat gatacaacac ca#ctagcagg   4200tttgagactc tcacaagttc gatcgatggc atttttcgga caagtcaagt gt#atgccttc   4260cattgcagat tataggcttt ttcgagttct gattctttgt ttttgggctg at#caagagaa   4320aacaagctat gacctcacaa gcatttttga actgttacaa ctgagatatc tg#aagataac   4380aggtaatatc acagttaaac ttccagagaa gatccaagga ctacaacact tg#cagacact   4440ggaagcagat gcaagagcaa ctgctgtcct attggatatt gttcatacac ag#tgtttgtt   4500gcaccttcgt cttgtactac ttgatctgct ccctcactgt cacaggtaca tc#ttcaccag   4560catccccaaa tggactggaa agctcaacaa tctccgcatt ttaaacattg ca#gtcatgca   4620aatttcccag gatgaccttg acactctcaa aggactggga tctctcactg ct#ctttcgct   4680gcttgttcga acagcgcctg cgcaaagaat cgtcgctgcg aatgaggggt tc#gggtctct   4740caagtacttc atgtttgtct gtacagcacc atgcatgact tttgtggaag ga#gcaatgcc   4800gagtgtgcaa aggttaaatc taaggttcaa tgccaacgag ttcaagcagt at#gactctaa   4860ggagacaggg ttggaacact tggtcgccct tgcagagatc tctgcaagaa tt#gggggcac   4920tgatgatgat gaatcaaaca aaactgaagt ggagtctgcc ttgaggactg ca#attcgcaa   4980gcatccgacg ccgagcactc ttatggttga tatacaatgg gtggattgga tc#tttggtgc   5040tgaagggaga gacttggatg aagatttggc acaacaagat gatcacgggt at#ggattttt   5100cattctattc ccaggttaca acttacaagg attattgagc ttctttcttt ct#ctgccgtg   5160gcttctatct ttacctgcta tgcatcttca acctgacttg atgattgttt ga#aaccaagc   5220ttcccatggt gacgtaccgg tagatctgac aaagcagcat tagtccgttg at#cggtggaa   5280gaccactcgt cagtgttgag ttgaatgttt gatcaataaa atacggcaat gc#tgtaaggg   5340ttgtttttta tgccattgat aatacactgt actgttcagt tgttgaactc ta#tttcttag   5400ccatgccaag tgcttttctt attttgaata acattacagc aaaaagttga tt#agactgtg   5460ttcggcgttc cccctaaatt tctcccccta tatctcactc acttgtcaca tc#agcgttct   5520ctttccccct atatctccac gctctacagc agttccacct atatcaaacc tc#tatacccc   5580accacaacaa tattatatac tttcatcttc aactaactca tgtaccttcc aa#ttttttct   5640actaataatt atttacgtgc acagaaactt aggcaaggga gagagagagc gg#tacc       5696

What is claimed is:
 1. An isolated nucleic acid fragment comprising anucleic acid sequence encoding an altered Pi-ta resistance polypeptideselected from the group consisting of: (a) a nucleic acid sequencehaving at least 90% sequence identity when compared to nucleotides2426-5212 of the nucleic acid sequence of SEQ ID NO:57, wherein thealtered Pi-ta resistance polypeptide has an amino acid substitution foralanine at position 918 and confers resistance in a plant transformedtherewith against the fungus Magnaporthe comprising in its genome one ormore virulent and/or avirulent AVR-Pita alleles, and (b) a nucleic acidsequence encoding an amino acid sequence as set forth in SEQ ID NO:58wherein the amino acid sequence has a single amino acid substitution atposition 918 which confers resistance in a plant transformed therewithagainst the fungus Magnaporthe comprising in its genome one or morevirulent and/or avirulent AVR-Pita alleles.
 2. The isolated nucleic acidfragment of claim 1 wherein the amino acid at position 918 is selectedfrom the group consisting of M, C, I, R, K, N, L and Q.
 3. A chimericgene comprising the nucleic acid fragment of claim 1 or 2 operablylinked to at least one regulatory sequence.
 4. A plant comprising in itsgenome the chimeric gene of claim
 3. 5. The plant of claim 4 whereinsaid plant is selected from the group consisting of rice, wheat, barley,corn, finger millet sorghum, and pearl millet.
 6. A transformed seed ofthe plant of claim
 4. 7. A method of conferring resistance in a plantagainst the fungus Magnaporthe comprising in its genome virulent and/oravirulent AVR-Pita alleles which comprises wherein said plant hasincreased resistance against the fungus Magnaporthe comprising in itsgenome one or more virulent and/or avirulent AVR-Pita alleles ascompared to an untransformed plant.
 8. A transformed seed of the plantof claim 5.